Fabric and home care products comprising cold water proteases

ABSTRACT

This invention relates to fabric and home care products comprising one or more cold water proteases and processes for making and using such products. Such compositions provide improved cleaning and freshness. Such cold water proteases may be derived from parent enzymes, including BPN′ subtilisin and subtilisin derived from  Bacillus lentus , by substitution, insertion and/or deletion of one or more of the parent enzymes&#39; amino acids.

FIELD OF INVENTION

This invention relates to fabric and home care products comprising coldwater proteases as well as methods of making and using such fabric andhome care products.

BACKGROUND OF THE INVENTION

Detergent manufacturers incorporate proteases into their products toprovide good cleaning of stains (such as blood). However, given thesustainability and consumer trends to lower wash temperatures it isproving increasingly difficult to deliver consumer acceptable benefitsand there remains a need to improve the cleaning and freshness profileof these laundry detergent compositions. The Inventors have found thatadditionally incorporating certain cold water proteases into a fabricand home care products, for example, a laundry detergent compositionthat comprises a hueing agent, a cleaning polymer and/or a perfumecapsule, improves the whiteness, whiteness perception, and/or freshness.

SUMMARY OF THE INVENTION

This invention relates to fabric and home care products comprising oneor more cold water proteases and processes for making and using suchproducts. Such compositions provide improved cleaning and freshness.Such cold water proteases may be derived from parent enzymes, includingBPN′ subtilisin and subtilisin derived from Bacillus lentus, bysubstitution, insertion and/or deletion of one or more of the parentenzymes' amino acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a plasmid map of pHPLT-BPN′-v3.

FIG. 2 provides a plasmid map of pHPLT-BPN′-v3+S78N.

FIG. 3 provides a plasmid map of pHPLT-BPN′ partial opt.

FIG. 4 provides a plasmid map of pHPLT-BPN′-v36.

FIG. 5 provides an alignment of the mature reference subtilisinproteases including: BPN′ (SEQ ID NO:2) and GG36 (SEQ ID NO:755). Eachamino acid position of each protease variant described herein, includingeach cold water protease variant, is numbered according to the numberingof the corresponding amino acid position in the amino acid sequence ofBacillus amyloliquefaciens subtilisin protease BPN′ (SEQ ID NO:2), asshown in FIG. 5, as determined by alignment of the protease variantamino acid sequence with the Bacillus amyloliquefaciens subtilisinprotease BPN′ amino acid sequence. Thus, unless otherwise specifiedherein, substitution positions are given in relationship to BPN′.

FIG. 6 provides map of pHPLT-GG36.

FIG. 7 provides a map of pRA68.

FIG. 8 provides a map of pRA96.

FIG. 9 provides a map of pAC-FNAre.

FIG. 10 is a schematic representation of method or targeted ISD libraryconstruction.

FIG. 11 provides a map of pAC-FNA10.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein “fabric and home products” means products or devicesgenerally intended to be used or consumed in the form in which they aresold and that are for treating fabrics, hard surfaces and any othersurfaces in the area of fabric and home care, including: air careincluding air fresheners and scent delivery systems, car care,dishwashing, fabric conditioning (including softening and/orfreshening), laundry detergency, laundry and rinse additive and/or care,hard surface cleaning and/or treatment including floor and toilet bowlcleaners, and other cleaning for consumer or institutional use.

As used herein, the term “cleaning and/or treatment composition” is asubset of fabric and home care products that includes, unless otherwiseindicated fabric & home care products. Such products include, but arenot limited to, products for treating fabrics, hard surfaces and anyother surfaces in the area of fabric and home care, including: air careincluding air fresheners and scent delivery systems, car care,dishwashing, fabric conditioning (including softening and/orfreshening), laundry detergency, laundry and rinse additive and/or care,hard surface cleaning and/or treatment including floor and toilet bowlcleaners, granular or powder-form all-purpose or “heavy-duty” washingagents, especially cleaning detergents; liquid, gel or paste-formall-purpose washing agents, especially the so-called heavy-duty liquidtypes; liquid fine-fabric detergents; hand dishwashing agents or lightduty dishwashing agents, especially those of the high-foaming type;machine dishwashing agents, including the various tablet, granular,liquid and rinse-aid types for household and institutional use: car orcarpet shampoos, bathroom cleaners including toilet bowl cleaners; aswell as cleaning auxiliaries such as bleach additives and “stain-stick”or pre-treat types, substrate-laden products such as dryer added sheets.

As used herein, the term “fabric and/or hard surface cleaning and/ortreatment composition” is a subset of cleaning and treatmentcompositions that includes, unless otherwise indicated, granular orpowder-form all-purpose or “heavy-duty” washing agents, especiallycleaning detergents; liquid, gel or paste-form all-purpose washingagents, especially the so-called heavy-duty liquid types; liquidfine-fabric detergents; hand dishwashing agents or light dutydishwashing agents, especially those of the high-foaming type; machinedishwashing agents, including the various tablet, granular, liquid andrinse-aid types for household and institutional use; liquid cleaning anddisinfecting agents, car or carpet shampoos, bathroom cleaners includingtoilet bowl cleaners; fabric conditioning products including softeningand/or freshening that may be in liquid, solid and/or dryer sheet form;as well as cleaning auxiliaries such as bleach additives and“stain-stick” or pre-treat types, substrate-laden products such as dryeradded sheets. All of such products which are applicable may be instandard, concentrated or even highly concentrated form even to theextent that such products may in certain aspect be non-aqueous.

As used herein, articles such as “a” and “an” when used in a claim, areunderstood to mean one or more of what is claimed or described.

As used herein, the terms “include”, “includes” and “including” aremeant to be non-limiting.

As used herein, the term “solid” includes granular, powder, bar andtablet product forms.

As used herein, the term “fluid” includes liquid, gel, paste and gasproduct forms.

As used herein, the term “situs” includes fabrics, garments, and/or hardsurfaces.

Unless otherwise noted, all component or composition levels are inreference to the active portion of that component or composition, andare exclusive of impurities, for example, residual solvents orby-products, which may be present in commercially available sources ofsuch components or compositions.

All percentages and ratios are calculated by weight unless otherwiseindicated. All percentages and ratios are calculated based on the totalcomposition unless otherwise indicated.

It should be understood that every maximum numerical limitation giventhroughout this specification includes every lower numerical limitation,as if such lower numerical limitations were expressly written herein.Every minimum numerical limitation given throughout this specificationwill include every higher numerical limitation, as if such highernumerical limitations were expressly written herein. Every numericalrange given throughout this specification will include every narrowernumerical range that falls within such broader numerical range, as ifsuch narrower numerical ranges were all expressly written herein.

Fabric and Home Products Comprising One or More Proteases

A composition comprising a protease, said protease being selected fromthe group consisting of:

-   a) a cold water protease having a performance index greater than 1,    at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least    1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at    least 2 from 1 to about 10, from 1 to about 8 or even from 1 to    about 5 on BMI at pH 8 and 60° F. when compared to an enzyme having    SEQ ID NO:4, as defined in Test Method 2 and/or Test Method 3;-   b) a cold water protease having a performance index at least 1, at    least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5,    at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2    from 1 to about 10, from 1 to about 8 or even from 1 to about 5 on    BMI at pH 8 and 60° F. when compared to an enzyme having SEQ ID    NO:6, as defined in Test Method 2 and/or Test Method 3;-   c) a cold water protease, said cold water protease being selected    from the group consisting of:    -   (i) a variant of a parent protease, said parent protease having        at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98%        or 99% or even 100% identity to SEQ ID NO:4, said variant        comprising a group of mutations selected from the following        groups of mutations: P210S, P210S-N218A,        S063T-S078N-S101A-S183T-T244N, N061A-S078N-S224A,        S053G-S078N-P129T-Q185T, S063T-S078N-S101A, S078N-P129T,        S063T-S078N-S101A-S183T and S063T-S078N-S101A-T2441;    -   (ii) a variant of a parent protease, said parent protease having        at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98%        or 99% or even 100% identity to SEQ ID NO:2, said variant        comprising a group of mutations selected from the following        groups of mutations: G097A-I111V-M124V-Y217Q,        G097A-I111V-Y167A-Y217Q,        S024G-N025G-N061P-G097A-S101N-G128S-Y217Q,        S024G-N025G-S053G-N061P-G097A-S101N-G128A-V203Y-Y217Q,        S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128S-V203Y-Y217Q,        V068A-A092G-Y217Q, N061P-G097A-S101N-G128A-P201S-Y217Q,        S024G-N025G-S053G-N061P-G097A-S101N-G128A-P210S-Y217Q,        S024G-N025G-S053G-N061P-G097A-S101N-G128S-Y217Q,        S024G-N025G-S053G-N061P-S078N-G097A-S101N-I111V-G128S-Y217Q,        S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,        N025G-G097A-S101N-G128A-Y217Q,        N025G-S038G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,        N025G-S053G-N061P-S078N-G128A-Y217Q,        N025G-S053G-N061P-S078N-S101N-G128A-Y217Q,        N025G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,        N025G-S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,        N025G-S078N-G097A-S101N-G128A-Y217Q,        N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,        N025G-T055P-N061P-S078N-S101N-G128A-Y217Q,        N061P-S101N-G128A-Y217Q, S024G-N025G-N061P-G097A-G128A-Y217Q,        S024G-N025G-N061P-G097A-S101N-G128A-Y217Q,        S024G-N025G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,        S024G-N025G-S053G-N061P-S078N-S101N-G128A-Y217Q,        S024G-N025G-S053G-T055P-G097A-S101N-G128A-Y217Q,        S024G-N025G-S053G-T055P-N061P-G128A-Y217Q,        S024G-N025G-T055P-G097A-G128A-Y217Q,        S024G-N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,        S024G-N061P-S078N-G097A-S101N-G128A-Y217Q,        S024G-S053G-N061P-G097A-G128A-Y217Q,        S024G-S053G-N061P-S078N-G097A-G128A-Y217Q,        S024G-S053G-T055P-G097A-S101N-G128A-Y217Q,        S024G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,        S024G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,        S024G-S053G-T055P-N061P-S101N-G128A-Y217Q,        S024G-T055P-N061P-G097A-G128A-Y217Q,        S053G-G097A-S101N-G128A-Y217Q,        S053G-N061P-G097A-S101N-G128A-Y217Q-S249N,        S053G-N061P-S078N-G097A-G128A-Y217Q,        S053G-S078N-G097A-S101N-G128A-Y217Q,        S053G-T055P-G097A-S101N-G128A-Y217Q,        S053G-T055P-N061P-S101N-G128A-Y217Q,        S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,        T055P-G097A-S101N-G128A-Y217Q,        T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,        S024G-N025G-N061P-S078N-G097A-S101N-G128A-Y217Q,        S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,        S024G-T055P-N061P-S078N-S101N-G128A-Y217Q,        T055P-N061P-S078N-G128A-Y217Q and        S24G-S53G-S78N-S101N-G128A-Y217Q;    -   (iii) a variant of a parent protease, said parent having at        least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98% or        99% or even 100% identity to SEQ ID NO:2, said variant        comprising three, four, five or six of the following mutations        X24G/R, X53G, X78N, X101N, X128A/S and X217L/Q; said variant        optionally comprising at least one group of mutations selected        from the following groups of mutations: A116V, G160S, I111L,        I115V, N109S, N117M, P005G, Q059V, T164S, Y262M, A015Q, A015S,        A098E, A098N, A098S, A098T, A098V, A098Y, A114S, A114T, A116G,        A116L, A116S, A116T, A116W, A133G, A133H, A133T, A133V, A137G,        A137I, A137L, A137S, A137T, A138S, A216E, A216F, A216V, D099S,        D181E, F261A, F261Q, G024F, G024I, G024Q, G024Y, G097S, G160T,        G211L, G211V, H017F, H017W, H039V, H226A, I031V, I111V, I268V,        K170R, K265R, L016Q, L016T, L135M, L209T, L209V, L233M, L257T,        L257V, L267A, L267V, N025A, N025I, N025Q, N025R, N025T, N025V,        N101I, N101Q, N101S, N109A, N109G, N109H, N109L, N109M, N109Q,        N109T, N117Q, N184A, N184L, N184T, N184W, N212G, N212L, N212V,        N243P, N252G, N252M, P005T, P014S, P040G, P040L, P040Q, P129A,        P129S, P172G, P172S, P194Q, P210A, P210S, Q185F, Q185G, Q185I,        Q185M, Q185N, Q185S, Q275H, R186K, S009A, S009G, S009H, S009M,        S018T, S130T, S132N, S145K, S159T, S161I, S161K, S161N, S161T,        S162I, S162M, S162Y, S163G, S182F, S182G, S182V, S182W, S183F,        S183L, S183M, S183T, S183V, S183W, S224A, S236T, S249V, T022A,        T022G, T022Q, T022V, T208V, T242S, T253N, T253S, T254A, T254S,        T255L, T255S, T255V, V004A, V004P, V004W, V084C, V139C, V165M,        V203F, Y021K, Y021N, Y021T, Y021V, Y167F, Y171F, Y214F, Y262F,        Y262T, A088T-L257G, A116T-A128S, N061S-N109G-A128S-N243V-S260P,        S009T-N109G-A128S-K141R-N243V,        S009T-S018T-Y021N-N109G-A128S-K141R, S162G-K256R, A088T-N243V,        G024E-A116T, K043Y, N076D-A116T, N218S-S248N, S033T-N243V,        S033T-S063G, S248N-L257G, A001E-S249A, A088T-A116T, A088T-A128S,        A088T-G131H, A088T-N109G, A088T-S248N, A088T-S249A, A116T-N243V,        A116T-T158S, A128S, A128S-K256R, A128S-L257G, A128S-N243V,        A128S-S248N, A128S-T158S, G024E-A088T, G024E-A128S, G024E-G131H,        G024E-K256R, G024E-L257G, G024E-N218S, G024E-N243V, G024E-S162G,        G024E-S249A, G024E-T158S, G131H, G131H-K256R, G131H-S249A,        K043Y-A088T, K043Y-A116T, K256R, N076D-K256R, N109G,        N109G-A116T, N109G-A128S, N109G-A128S-N243V-K256R,        N109G-A128S-N243V-S248A, N109G-G131H, N109G-K256R, N109G-L257G,        N109G-N218S, N109G-N243V, N109G-S248N, N218S-L257G, N243V,        N243V-K256R, N243V-L257G, N243V-S248N, N243V-S249A, Q103H-A128S,        Q103H-G131H, Q103H-K256R, Q103H-L257G, Q103H-N243V, Q103H-S248N,        Q103H-S249A, Q103H-T158S, Q206D-N243V, S033T-A128S, S033T-K256R,        S033T-N076D, S033T-N218S, S033T-S248N, S033T-T158S, S063G-A128S,        S063G-K256R, S063G-N243V, S063G-S162G, S063G-T158S, S248N-K256R,        S249A, T158S-N243V, T158S-S249A, A088T-A116T-N243V-K256R-L257G,        A088T-A116T-N243V-L257G, A088T-T158S-N218S-K256R,        A088T-T158S-N218S-N243V-L257G,        A088T-A116T-T158S-N218S-N243V-K256R-L257G,        A088T-N109G-A116T-G131H-A153S-N218S-S248N-L257G,        A088T-N109G-A116T-T158S-S248N-K256R-L257G,        A088T-N109G-T158S-L257G,        A114S-A116T-N218S-N243V-S248N-K256R-L257G, A116T-T158S-K256R,        A088T-A116T-G131H-T158S-S248N-L257G, A088T-A116T-T158S,        A088T-N109G-A116T-G131H-L257G,        A088T-N109G-A116T-T158S-N243V-S248N-L257G,        A088T-N109G-N243V-L257G, A088T-N109G-N243V-S248N,        A088T-N109G-T158S-N243V-L257G,        A088T-N109G-T158S-N243V-S248N-L257G, A116T-T158S-S248N-L257G,        Y006H-A116T-G131H-S248N, A088T-A116T-G131H-T158S-N218S-N243V,        A088T-A116T-G131H-T158S-N243V,        A088T-A116T-G131H-T158S-N243V-K256R-L257G,        A088T-A116T-N218S-N243V-K256R-L257G,        A088T-A116T-S248N-K256R-L257G, A088T-A116T-T158S-N218S-N243V,        A088T-A116T-T158S-N243V-K256R-L257G,        A088T-A116T-T158S-N243V-S248N-L257G,        A088T-G131H-N243V-S248N-K256R-L257G,        A088T-N109G-A116T-T158S-L257G,        A088T-N109G-A116T-T158S-N212D-N243V-K256R-L257G,        A088T-N109G-A116T-T158S-N218S-N243V-S248N-K256R,        A088T-N109G-A116T-T158S-S248N-L257G,        A088T-N109G-G131H-V148A-N218S-N243V-K256R-L257G,        A088T-N109G-K256R, A088T-N109G-N243V-S248N-L257G,        A088T-N109G-T158S-K256R A088T-N109G-T158S-N243V,        A088T-T158S-N243V-K256R-L257G, A116T-N218S-N243V-L257G-N269S,        A116T-T158S-K256R-L257G, N109G-A116T-K256R-L257G,        N109G-A116T-N243V, N109G-A116T-T158S-N243V-K256R-L257G,        N109G-G131H-L257G, N109G-G131H-S248N-K256R-L257G,        N109G-G131H-T158S-K256R-L257G, S003P-A116T-T158S-S248N-K256R,        T158S-S248N-K256R, A088T-A116T-G131H-N243V-K256R,        A088T-A116T-G131H-S248N-K256R-L257G,        A088T-A116T-G131H-V147A-T158S-N218S-N243V-S248N-L257G,        A088T-A116T-S248N-L257G, A088T-A116T-T158S-N218S,        A088T-A116T-T158S-N218S-K256R-L257G,        A088T-A116T-T158S-N218S-L257G, A088T-G131H-N243V-L257G,        A088T-G131H-T158S-S248N-L257G, A088T-N109G-A116T,        A088T-N109G-A116T-G131H-N218S,        A088T-N109G-A116T-G131H-N218S-S248N-L257G,        A088T-N109G-A116T-G131H-N243V-S248N-K256R-L257G,        A088T-N109G-A116T-G131H-T158S-S248N-K256R-L257G,        A088T-N109G-A116T-N218S-N243V-K256R,        A088T-N109G-A116T-N218S-N243V-L257G,        A088T-N109G-A116T-N243V-S248N-K256R,        A088T-N109G-A116T-N243V-S248N-K256R-L257G,        A088T-N109G-A116T-T158S-N243V-L257G,        A088T-N109G-G131H-T158S-N243V-S248N-K256R,        A088T-N109G-G131H-T158S-W241R-S248N-K256R,        A088T-N109G-K256R-L257G, A088T-N109G-L257G, A088T-N109G-N243V,        A088T-N109G-N243V-K256R, A088T-N109G-N243V-K256R-L257G,        A088T-N109G-S248N-K256R, A088T-N109G-T158S-N218S-K256R-L257G,        A088T-N109G-T158S-N218S-N243V-S248N-K256R,        A088T-N109G-T158S-N243V-K256R,        A088T-N109G-T158S-N243V-K256R-L257G,        A088T-N109G-T158S-N243V-S248N-A274D,        A088T-N109G-T158S-S248N-L257G, A088T-T158S-K256R,        A088T-T158S-N218S-N243V-K256R-L257G, A088T-T158S-N243V-L257G,        A116T-G131H-N218S-N243V-S248N, A116T-G131H-S248N-L257G,        A116T-S248N-K256R-L257G, A116T-T158S-N218S-N243V-K256R,        A116T-T158S-N218S-S248N-L257G-Q271R,        A116T-T158S-N243V-K256R-L257G, A116T-T158S-N243V-S248N-L257G,        G131H-S248N, G131H-T158S-I234T-N243V-K256R,        G131H-W241L-N243V-S248N-K256R,        N109G-A116T-G131H-A137V-T158S-S248N-K256R-L257G,        N109G-A116T-G131H-A151S-N218S-K256R-L257G,        N109G-A116T-G131H-T158S-N218S-N243V-K256R,        N109G-A116T-G131H-T158S-N218S-S248N,        N109G-A116T-G131H-T158S-N243V-S248N, N109G-A116T-S248N,        N109G-A116T-T158S-L257G, N109G-A116T-T158S-N218S-W241R-N243V,        N109G-A116T-T158S-N243V-S248N-L257G,        N109G-A116T-T158S-S248N-K256R-L257G,        N109G-A116T-T158S-S248N-L257G, N109G-G131H-N218S-L257G,        N109G-G131H-N218S-S248N-K256R-L257G,        N109G-G131H-T158S-N218S-S248N-K256R-L257G-A274T,        N109G-N243V-L257G, N109G-T158S-N218S-K256R-L257G,        N109G-T158S-N218S-L257G, N109G-T158S-S248N-K256R,        P014L-A015L-L016C-H017T-S018L-Q019K-G020A-Y021T-T022L-G023E,        S003F-A088T-N109G-A116T-T158S-N243V-K256R-L257G,        V004A-A088T-A116T-T158S-N218S,        V004A-N109G-A116T-G131H-S248N-K256R-L257G,        V004L-A116T-N218S-N243V-S248N-L257G,        Y006H-N109G-N218S-N243V-S248N, A001T-A116T-T158S-N243V-L257G,        A088T-A116T-G131H-L257G, A088T-A116T-G131H-N218S-L257G,        A088T-A116T-G131H-N218S-S248N-K256R-L257G,        A088T-A116T-G131H-N218S-S248N-L257G,        A088T-A116T-G131H-N243V-K256R-L257G,        A088T-A116T-G131H-N243V-L257G, A088T-A116T-G131H-N243V-S248N,        A088T-A116T-G131H-T158S-K256R-L257G,        A088T-A116T-G131H-T158S-L257G, A088T-A116T-G131H-T158S-N218S,        A088T-A116T-G131H-T158S-N218S-N243V-K256R-A273T,        A088T-A116T-G131H-T158S-N218S-S248N-K256R,        A088T-A116T-G131H-T158S-N218S-S248N-L257G,        A088T-A116T-G131H-T158S-N243V-S248N-K256R,        A088T-A116T-G131H-T158S-S248N, A088T-A116T-K256R,        A088T-A116T-K256R-L257G, A088T-A116T-N218S-N243V-L257G,        A088T-A116T-N243V-K256R, A088T-A116T-N243V-S248N-K256R-L257G,        A088T-A116T-S248N-K256R, A088T-A116T-T158S-K256R,        A088T-A116T-T158S-N218S-N243V-K256R,        A088T-A116T-T158S-N218S-N243V-K256R-N269S,        A088T-A116T-T158S-N218S-N243V-S248N,        A088T-A116T-T158S-N218S-N243V-S248N-K256R-L257G,        A088T-A116T-T158S-N243V-K256R, A088T-A116T-T158S-N243V-L257G,        A088T-A116T-T158S-N243V-S248N-K256R,        A088T-A116T-T158S-N243V-S248N-K256R-L257G,        A088T-A116T-T158S-S248N-K256R,        A088T-A116T-V143A-N218S-S248N-K256R,        A088T-A116T-V147I-T158S-N218S-N243V-L257G,        A088T-G131H-K256R-L257G, A088T-G131H-N218S-N243V-S248N,        A088T-G131H-N218S-S248N-L257G, A088T-G131H-S248N-K256R-L257G,        A088T-G131H-T158S-L257G, A088T-G131H-T158S-N218S-K256R,        A088T-G131H-T158S-N218S-N243V-K256R-L257G,        A088T-G131H-T158S-N218S-N243V-L257G,        A088T-G131H-T158S-N218S-S248N, A088T-G131H-T158S-N243V,        A088T-G131H-T158S-N243V-S248N,        A088T-G131H-T158S-N243V-S248N-K256R,        A088T-G131H-T158S-N243V-S248N-L257G,        A088T-I107T-N109G-A116T-G131H-T158S-N218S-N243V-S248N,        A088T-N109G-A116T-G131H-N218S-L257G,        A088T-N109G-A116T-G131H-N218S-N243V,        A088T-N109G-A116T-G131H-N218S-N243V-K256R-L257G,        A088T-N109G-A116T-G131H-N218S-N243V-L257G,        A088T-N109G-A116T-G131H-N218S-S248N-K256R-L257G,        A088T-N109G-A116T-G131H-N243V,        A088T-N109G-A116T-G131H-N243V-L257G,        A088T-N109G-A116T-G131H-N243V-S248N-L257G,        A088T-N109G-A116T-G131H-S248N,        A088T-N109G-A116T-G131H-S248N-K256R,        A088T-N109G-A116T-G131H-S248N-L257G,        A088T-N109G-A116T-G131H-T158S-L257G,        A088T-N109G-A116T-G131H-T158S-N218S,        A088T-N109G-A116T-G131H-T158S-N218S-S248N-K256R,        A088T-N109G-A116T-G131H-T158S-N218T-N243V,        A088T-N109G-A116T-G131H-T158S-N243V-K256R,        A088T-N109G-A116T-G131H-T158S-N243V-K256R-L257G,        A088T-N109G-A116T-G131H-T158S-N243V-S248N,        A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R,        A088T-N109G-A116T-G131H-T158S-S248N-K256R-L257G,        A088T-N109G-A116T-G131H-T158S-S248N-L257G,        A088T-N109G-A116T-N218S, A088T-N109G-A116T-N218S-L257G,        A088T-N109G-A116T-N218S-N243V,        A088T-N109G-A116T-N218S-N243V-S248N-L257G,        A088T-N109G-A116T-N218S-S248N-K256R,        A088T-N109G-A116T-N218T-K256R,        A088T-N109G-A116T-N218T-K256R-L257G, A088T-N109G-A116T-N243V,        A088T-N109G-A116T-N243V-K256R-L257G,        A088T-N109G-A116T-N243V-K256R-L257G-N269D,        A088T-N109G-A116T-S248N-K256R, A088T-N109G-A116T-T158S,        A088T-N109G-A116T-T158S-N218S-L257G,        A088T-N109G-A116T-T158S-N218S-N243V,        A088T-N109G-A116T-T158S-N218S-N243V-K256R,        A088T-N109G-A116T-T158S-N218S-N243V-K256R-L257G,        A088T-N109G-A116T-T158S-N218S-N243V-L257G,        A088T-N109G-A116T-T158S-N218S-S248N,        A088T-N109G-A116T-T158S-N243V,        A088T-N109G-A116T-T158S-N243V-K256R,        A088T-N109G-A116T-T158S-N243V-K256R-L257G,        A088T-N109G-G131H-L257G, A088T-N109G-G131H-N218S-K256R-L257G,        A088T-N109G-G131H-N218S-N243V-K256R,        A088T-N109G-G131H-N218S-N243V-L257G,        A088T-N109G-G131H-N218S-N243V-S248N-K256R-L257G,        A088T-N109G-G131H-N243V, A088T-N109G-G131H-N243V-L257G,        A088T-N109G-G131H-N243V-S248N-K256R,        A088T-N109G-G131H-N243V-S248N-L257G,        A088T-N109G-G131H-S248N-L257G, A088T-N109G-G131H-T158S-L257G,        A088T-N109G-G131H-T158S-N218S-N243V-S248N-K256R,        A088T-N109G-G131H-T158S-N243V,        A088T-N109G-G131H-T158S-N243V-K256R,        A088T-N109G-G131H-T158S-N243V-K256R-L257G,        A088T-N109G-G131H-T158S-N243V-L257G, A088T-N109G-N218S-K256R,        A088T-N109G-N218S-N243V-S248N-L257G,        A088T-N109G-N218S-S248N-K256R-L257G,        A088T-N109G-N243V-S248N-K256R-L257G,        A088T-N109G-N243V-S248N-L257G-I268V,        A088T-N109G-S248N-K256R-L257G, A088T-N109G-T158S-N218S-K256R,        A088T-N109G-T158S-N218S-N243V-L257G,        A088T-N109G-T158S-N243V-K256R-I268V,        A088T-N109G-T158S-N243V-S248N-Q275R, A088T-N218S-N243V,        A088T-N218S-N243V-S248N-K256R-L257G, A088T-N218S-S248N,        A088T-N218S-S248N-L257G, A088T-N243V-K256R, A088T-N243V-L257G,        A088T-S145T-T158S-S248N, A088T-T158S-L257G,        A088T-T158S-N218S-S248N-L257G,        A088T-T158S-N243V-K256R-L257G-Q271H, A088T-T158S-S248N,        A088T-V143A-T158S-K256R, A116T-G131H-K256R, A116T-G131H-N218S,        A116T-G131H-N243V, A116T-G131H-N243V-K256R,        A116T-G131H-N243V-L257G, A116T-G131H-S248N-K256R,        A116T-G131H-T158S-N218S-I234T-N243V-S248N-K256R,        A116T-G131H-T158S-N243V-L257G,        A116T-G131H-T158S-N243V-S248N-K256R,        A116T-G131H-V143F-T158S-N218S, A116T-L257G, A116T-N218S,        A116T-N218S-L257G, A116T-N218S-N243V-L257G, A116T-N243V-K256R,        A116T-N243V-S248N, A116T-N243V-S248N-K256R-L257G, A116T-S248N,        A116T-T158S-N218S-N243V, A116T-T158S-N218S-S248N,        A116T-T158S-N243V, A116T-T158S-N243V-K256R,        A116T-T158S-N243V-L257G, A116T-T158S-N243V-S248N,        A116T-T158S-S248N-K256R-L257G,        A116T-V149I-T158S-N243V-S248N-K256R-Q271H,        G131H-N218S-N243V-L257G, G131H-N243V, G131H-N243V-S248N-K256R,        G131H-T158S, G131H-T158S-N218S-N243V-K256R,        G131H-T158S-N243V-K256R-L257G, G131H-T158S-N243V-S248N-L257G,        N109G-A116T-G131H-N218S-K256R-L257G,        N109G-A116T-G131H-N218S-L257G,        N109G-A116T-G131H-N218S-N243V-K256R-L257G,        N109G-A116T-G131H-N218S-S248N-K256R,        N109G-A116T-G131H-N243V-K256R, N109G-A116T-G131H-N243V-L257G,        N109G-A116T-G131H-N243V-S248N-K256R-L257G,        N109G-A116T-G131H-S248N, N109G-A116T-G131H-S248N-I268V,        N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R,        N109G-A116T-G131H-T158S-N218S-S248N-L257G,        N109G-A116T-G131H-T158S-S248N,        N109G-A116T-G131H-T158S-S248N-K256R, N109G-A116T-N218S,        N109G-A116T-N218S-N243V-K256R,        N109G-A116T-N218S-N243V-K256R-L257G,        N109G-A116T-N218S-S248N-L257G, N109G-A116T-N243V-K256R,        N109G-A116T-N243V-S248N-K256R-L257G, N109G-A116T-S248N-L257G,        N109G-A116T-T158S-G211V-N243V-S248N-K256R,        N109G-A116T-T158S-K256R-L257G, N109G-A116T-T158S-N218S,        N109G-A116T-T158S-N218S-N243V-K256R-L257G,        N109G-A116T-T158S-N218S-N243V-L257G,        N109G-A116T-T158S-N218S-N243V-S248N-L257G,        N109G-A116T-T158S-N218S-S248N-K256R-L257G,        N109G-A116T-T158S-N243V, N109G-A116T-T158S-Q275R,        N109G-G131H-A137V-T158S-N218S-S248N, N109G-G131H-N218S-K237N,        N109G-G131H-N218S-N243V-K256R-L257G,        N109G-G131H-N218S-S248N-K256R, N109G-G131H-N243V-K256R-L257G,        N109G-G131H-S145F-N218S-N243V-K256R-L257G,        N109G-G131H-S248N-K256R, N109G-G131H-S248N-L257G,        N109G-G131H-T158S-K256R, N109G-G131H-T158S-N218S-N243V-K256R,        N109G-G131H-T158S-N243V, N109G-G131H-T158S-N243V-K256R-L257G,        N109G-G131H-T158S-N243V-L257G, N109G-G131H-T158S-S248N-L257G,        N109G-G131H-T158S-S248N-Q271R, N109G-N218S-L257G,        N109G-N218S-N243V, N109G-N243V-K256R-L257G,        N109G-N243V-S248N-K256R-L257G, N109G-T158S-I268V,        N109G-T158S-K256R, N109G-T158S-N218S-N243V-K256R-L257G,        N109G-T158S-N218S-S248N-L257G, N109G-T158S-N243V,        N109G-T158S-N243V-K256R-L257G, N109G-T158S-N243V-S248N,        N109S-A116T-S248N, N218S, N218S-N243V-S248N-K256R-L257G,        N218S-S248N-L257G, N243V-K256R, N243V-S248N-K256R,        N243V-S248N-K256R-L257G,        S105P-A116T-T158S-N218S-N243V-S248N-K256R, S248N,        T158S-N243V-K256R, T158S-N243V-L257G, S018F-S162L, S018P-D120N,        P014T-S037T, S009T-K141R, S161P-S162L,        N61P-S63G-N109Q-A128S-S224A-N243V,        A88T-N109G-A114S-A116T-A128S-N243V,        A88T-N109G-A114S-A116T-A128S-S183L-S224A-N243V,        N109G-A128S-S183V, N109M-A128S-S224A,        A88T-N109S-A116T-A128S-S224A-N243V, N109Q-A128S-S224A-N243V,        A88T-N109M-A116T-A128S-S224A-N243V, S63G-A128S,        N109S-A128S-S224A-N243V, A88T-N109G-A116T-N243V,        N61S-N109G-N243V, N101Q-N109Q-A128S-S224A-N243V,        A88T-N109G-A116T-T158S-N243V-K256R, N109G-A116T, A88T-N109G,        N61G-N109G-N243V, N109G-A128S-P129S-S130T-S224A-N243V,        A88T-N109Q-A116T-A128S-S224A-N243V, N062L-S063N-Q217L,        N062S-S063R-Q217E, N062S-S063L-Q217L, S063G, S063G-Q217L, S063N        and S063N-Q217L;-   d) a cold water protease, said cold water protease being selected    from the group consisting of:    -   (i) a variant of a parent protease, said parent protease having        at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98%        or 99% or even 100% identity to SEQ ID NO:4, said variant        comprising a group of mutations selected from the following        groups of mutations: E156S-P210S, P210S-N218S,        Y104F-E156S-P210I, E156A-P210S-N218S, N218A,        N061A-S078N-S087E-S224A, Q059V-S078N-G211A, Q059V-S078N-V147Q,        Q059V-S078N, Q059V-S078N-I108V-N252Q, S053G-S078N-P129T,        S078N-G211A, S078N-Q185T, S078N-V147Q, S078N, S078N-S224A,        S078N-S224A-A274D, S078N-I108V-V147Q, N061A-S078N-S224A,        S053G-S078N-P129T-Q185T and S078N-P129T;    -   (ii) a variant of a parent protease, said parent protease having        at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98%        or 99% or even 100% identity to SEQ ID NO:2, said variant        comprising a group of mutations selected from the following        groups of mutations: G097A-I111V-Y217Q, I111V-G128A-Y217Q,        I111V-M124V-Y167A, I111V-M124V-Y217Q, L096T-G097A-Y217Q,        N062Q-G097A-I111V, S053G-N061P-G097A-S101N-G128S-V203Y-Y217Q,        S089Y-M124V-Y217Q, V068A-I111V-Y217Q, G097A-I111V-M124V,        G097A-L126A-Y217Q, G097A-M124V-Y217Q, I111V-Y167A-Y217Q,        M124V-Y167A-Y217Q, P052L-V068A-G097A, S089Y-I111V-M124V,        V068A-A092G-G097A, V068A-A092G-I111V, V068A-G097A-I111V,        V068A-S089Y-I111V, Y104N-G128A-Y217Q, G097A-G128A-P210S-Y217Q,        G097A-G128S-P210S-Y217Q, G097A-I111V-M124I-Y217Q,        G097A-I111V-M124V-P210S-Y217Q, G097A-N123Q-P210S-Y217Q,        G097A-N123Q-Y217Q, N061P-G097A-G128A-P210S-Y217Q,        N061P-G097A-G128S-Y217Q, N061P-G097A-I111V-M124V-Y217Q,        N061P-G097A-N123Q-Y217Q, N061P-G097A-S101N-I111V-M124V-Y217Q,        N061P-G097A-S101N-N123Q-Y217Q, N061P-G102A-P129S-Y217Q,        N061P-N062Q-G097A-G100N-S101N-Y217Q,        N061P-N062Q-G097A-G100N-Y217Q,        N061P-N062Q-G097A-G100Q-P201S-Y217Q,        N061P-N062Q-G097A-I111V-Y217Q,        N061P-N062Q-G097A-S101N-I111V-Y217Q,        N061P-S078N-G097A-I111V-M124I-Y217Q,        N061P-S078N-G102A-I111V-P129S-Y217Q,        N062Q-G097A-I111V-P210S-Y217Q, N062Q-G097A-I111V-Y217Q,        N062Q-S078N-G097A-I111V-Y217Q,        S024G-N025G-N061P-G097A-S101N-G128A-P210S-Y217Q,        S024G-N025G-S053G-N061P-G097A-S101N-I111V-M124V-Y217Q,        S024G-N025G-S053G-N061P-G097A-S101N-N123Q-Y217Q,        S024G-N025G-S053G-N061P-N062Q-G097A-G100N-S101N-Y217Q,        S024G-N025G-S053G-N061P-N062Q-G097A-S101N-I111V-Y217Q,        S024G-N025G-S053G-N061P-S101N-G102A-P129S-Y217Q,        S053G-N061P-G097A-G128S-Y217Q, S053G-N061P-G097A-M124I-Y217Q,        S053G-N061P-G097A-S101N-I111V-M124V-Y217Q,        S053G-N061P-G102A-P129S-P210S-Y217Q,        S053G-N061P-G102A-P129S-Y217Q,        S053G-N061P-N062Q-G097A-G100N-S101N-Y217Q,        S053G-N061P-N062Q-G097A-S101N-I111V-Y217Q,        S053G-N061P-S101N-G102A-P129S-Y217Q,        S053G-S078N-G097A-I111V-G128S-Y217Q, S078N-G097A-G128S-Y217Q,        S078N-G097A-I111V-M124V-Y217Q, N061P-G097A-M124I-Y217Q,        N061P-G097A-M124V-Y217Q, N061P-N062Q-G097A-G100D-Y217Q,        N061P-N062Q-G097A-G100Q-S101N-Y217Q,        N061P-N062Q-G097A-G100Q-Y217Q, N061P-N062Q-G100N-G102A-Y217Q,        N061P-N062Q-S078N-G097A-G100N-I111V-Y217Q,        S024G-N025G-S053G-N061P-G097A-S101N-M124I-Y217Q,        S053G-N061P-G097A-S101N-M124I-Y217Q,        S053G-N061P-G097A-S101N-N123Q-Y217Q,        N025G-S078N-G097A-G128A-Y217Q, N025G-T055P-G097A-G128A-Y217Q,        S024G-G097A-S101N-G128A-Y217Q,        S024G-I035V-T055P-N061P-S078N-G097A-Y217Q,        S024G-N025G-N061P-S078N-G097A-S101N-G128A,        S024G-N025G-N061P-S078N-G097A-S101N-G128A-Y217Q,        S024G-N025G-N061P-S078N-S101N-G128A-Y217Q,        S024G-N025G-S053G-N061P-G097A-G128A-S130G-Y217Q,        S024G-N025G-S053G-N061P-G128A-Y217Q,        S024G-N025G-S053G-T055P-G097A-G128A-Y217Q,        S024G-N025G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q,        S024G-N025G-S053G-T055P-N061P-S078N-G128A-Y217Q,        S024G-N025G-S053G-T055P-S078N-S101N-G128A-Y217Q,        S024G-N025G-S053G-T055P-S101N-G128A-Y217Q,        S024G-N025G-T055P-G097A-S101N-G128A-Y217Q,        S024G-N025G-T055P-N061P-S078N-G097A-Y217Q,        S024G-N061P-G097A-S101N-G128A-Y217Q,        S024G-S038G-S053G-S078N-S101N-G128A-Y217Q,        S024G-S053G-S078N-G097A-S101N-G128A-Y217Q,        S024G-S053G-S078N-S101N-G128A-Y217Q,        S024G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q,        S024G-T055P-G097A-G128A-Y217Q,        S024G-T055P-N061P-G097A-S101N-G128A,        S024G-T055P-N061P-S078N-S101N-G128A-Y217Q,        S024G-T055P-S078N-G097A-S101N-G128A-Y217Q, S101N-G128A-Y217Q,        T055P-N061P-G097A-A116S-G128A, T055P-N061P-S078N-G128A-Y217Q,        N025G-G097A-S101N-G128A-Y217Q,        N025G-S038G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,        N025G-S053G-N061P-S078N-G128A-Y217Q,        N025G-S053G-N061P-S078N-S101N-G128A-Y217Q,        N025G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,        N025G-S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,        N025G-S078N-G097A-S101N-G128A-Y217Q,        N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,        N025G-T055P-N061P-S078N-S101N-G128A-Y217Q,        N061P-S101N-G128A-Y217Q, S024G-N025G-N061P-G097A-G128A-Y217Q,        S024G-N025G-N061P-G097A-S101N-G128A-Y217Q,        S024G-N025G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,        S024G-N025G-S053G-T055P-G097A-S101N-G128A-Y217Q,        S024G-N025G-S053G-T055P-N061P-G128A-Y217Q,        S024G-N025G-T055P-G097A-G128A-Y217Q,        S024G-N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,        S024G-N061P-S078N-G097A-S101N-G128A-Y217Q,        S024G-S053G-N061P-G097A-G128A-Y217Q,        S024G-S053G-N061P-S078N-G097A-G128A-Y217Q,        S024G-S053G-T055P-G097A-S101N-G128A-Y217Q,        S024G-S053G-T055P-N061P-S101N-G128A-Y217Q,        S024G-T055P-N061P-G097A-G128A-Y217Q,        S053G-N061P-G097A-S101N-G128A-Y217Q-S249N,        S053G-N061P-S078N-G097A-G128A-Y217Q,        S053G-S078N-G097A-S101N-G128A-Y217Q,        S053G-T055P-G097A-S101N-G128A-Y217Q,        S053G-T055P-N061P-S101N-G128A-Y217Q,        S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,        T055P-G097A-S101N-G128A-Y217Q and        T055P-N061P-S078N-G097A-S101N-G128A-Y217Q;    -   (iii) a variant of a parent protease, said parent protease,        having at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97%        or 98% or 99% or even 100% identity to SEQ ID NO:2, said variant        comprising three, four, five or six of the following mutations        X24G/R, X53G, X78N, X101N, X128A/S and X217L/Q, said variant        optionally comprising at least one group of mutations selected        from the following groups of mutations: A001G, A001Y, A013G,        A013V, A015F, A015G, A015K, A015M, A015P, A015T, A015W, A015Y,        A029G, A073S, A088C, A088I, A088L, A088T, A088V, A098D, A098K,        A098P, A098R, A098W, A116D, A116E, A116R, A128S, A133L, A133M,        A133S, A134G, A134S, A137N, A137V, A144M, A144Q, A144S, A144T,        A144V, A151C, A176S, A176T, A179S, A216G, A216L, A216P, A216Q,        A216S, A216T, A216Y, A228T, A230C, A231C, A272I, A272L, A272Q,        A272S, A272T, A272W, A273S, A274G, A274L, A274Q, A274T, A274V,        D041E, D099G, D099N, D120A, D120K, D120Q, D120R, D120S, D140E,        D181S, D259E, E054D, E156D, E156T, E251V, F261G, F261H, F261L,        F261R, F261S, F261T, F261V, F261W, G007A, G007S, G020A, G020D,        G020S, G024N, G024R, G024S, G024T, G024V, G024W, G053H, G053K,        G053N, G053T, G097A, G097D, G097T, G131A, G131H, G131P, G131Q,        G131T, G131V, G160H, G160P, G166A, G166S, G166T, G211A, G211D,        G211M, G211N, G211P, G211Q, G211R, G211W, G215A, G215N, G215V,        H017L, H017M, H017T, H017V, H017Y, H039A, H039C, H039N, H226F,        H226I, H226M, H226S, H226V, H226Y, I035V, I079A, I079S, I079T,        I079V, I079W, I108V, I115L, I122A, I234L, I234V, K012R, K027R,        K136R, K141F, K213W, K237R, K256R, K265Q, L016A, L016F, L016I,        L016S, L016V, L042I, L075A, L075M, L075Q, L075V, L075Y, L082K,        L082M, L082Q, L082V, L090I, L196I, L196V, L209Q, L209W, L233A,        L233Q, L233V, L235I, L235K, L250I, L257A, L257H, L257Q, L257S,        L257Y, L267Q, L267R, L267S, L267T, M199V, N025F, N025G, N025H,        N025K, N025L, N025M, N025S, N025Y, N061F, N061H, N061P, N061S,        N061T, N061V, N061W, N076G, N076W, N078S, N078T, N078V, N101A,        N101H, N101L, N101T, N109K, N109R, N117A, N117E, N117H, N117K,        N118G, N184G, N184H, N184I, N184S, N184V, N212A, N212F, N212I,        N212K, N212P, N212Q, N212S, N212Y, N218A, N218H, N218L, N218S,        N240E, N240H, N240L, N240R, N240T, N243A, N243Q, N243T, N243V,        N252A, N252K, N252L, N252Q, N252R, N252S, N252T, N269Q, N269S,        P014G, P014Q, P014T, P040A, P040H, P040S, P040T, P040V, P040Y,        P086A, P086C, P086F, P086H, P086S, P129D, P129G, P129K, P129T,        P172A, P172Q, P194A, P194G, P194L, P194M, P194S, P194V, P194Y,        P210G, P210R, P210V, Q002A, Q002S, Q010A, Q010F, Q010H, Q010I,        Q010L, Q010N, Q010S, Q010T, Q019A, Q019G, Q019N, Q019S, Q019T,        Q019V, Q019W, Q059I, Q103L, Q103S, Q185A, Q185H, Q185L, Q185T,        Q185Y, Q206P, Q206S, Q206Y, Q217I, Q217N, Q217S, Q217T, Q245K,        Q275D, Q275S, Q275W, S003A, S003G, S003H, S003M, S003P, S003Q,        S003T, S003V, S009I, S009L, S009P, S009T, S009W, S018A, S018G,        S018I, S018L, S018M, S018N, S018P, S018V, S018W, S033T, S037Q,        S037T, S037V, S038G, S038H, S038K, S038Q, S038T, S063K, S063N,        S063Q, S063T, S087A, S087F, S087G, S087Q, S087T, S089L, S089M,        S089N, S089Q, S089T, S089W, S130A, S130F, S130G, S130L, S130V,        S145A, S145H, S145M, S145V, S159A, S159G, S159H, S159Q, S159R,        S161A, S161G, S161H, S161L, S161M, S161P, S161Q, S161W, S162A,        S162F, S162G, S162L, S162N, S162P, S162R, S162V, S163P, S173A,        S173G, S182A, S182H, S182K, S182L, S182N, S182P, S182Q, S182T,        S183A, S183G, S183H, S183Q, S188A, S188G, S188T, S188V, S191A,        S204A, S204I, S204L, S204Q, S204V, S224C, S236A, S236N, S236Q,        S248A, S248F, S248G, S248I, S248K, S248L, S248M, S248N, S248Q,        S248T, S248V, S249A, S249C, S249H, S249Q, S249T, S249W, S249Y,        S260H, S260N, S260P, S260T, T022H, T022K, T022N, T022R, T022S,        T022Y, T055A, T055G, T055L, T055N, T055P, T055Q, T071S, T158H,        T158S, T164N, T208C, T208L, T220S, T242N, T244A, T244G, T244H,        T244I, T244Q, T244S, T244V, T244W, T253A, T253G, T253H, T253Q,        T254V, T255A, T255G, T255H, T255I, T255Q, T255Y, V004G, V004N,        V004R, V008A, V008C, V008M, V026I, V044I, V044L, V045H, V045K,        V045L, V045M, V045Q, V045S, V045W, V045Y, V051I, V081L, V081Q,        V081T, V084A, V084S, V084T, V093I, V121I, V143N, V143S, V143Y,        V147C, V147I, V147L, V147T, V180I, V180L, V180T, V192A, V192S,        V192T, V198I, V198L, V198M, V203H, V203I, V203L, V203N, V203Q,        V203T, V203W, V203Y, V270A, V270S, V270T, W241M, W241Y, Y006G,        Y006H, Y006I, Y006K, Y006L, Y006P, Y006Q, Y006T, Y006V, Y006W,        Y021A, Y021D, Y021E, Y021L, Y021Q, Y021R, Y021S, Y104F, Y104I,        Y214L, Y214V, Y214W, Y262A, Y262G, Y262L, Y262N, Y262S, Y262W,        Y263G, Y263W, A001F, A001K, A001L, A001M, A001Q, A001R, A001S,        A001T, A001V, A013C, A013S, A015D, A015E, A015L, A015R, A048S,        A073N, A073T, A074G, A074S, A085C, A085G, A085S, A085V, A088M,        A088S, A092S, A098G, A114G, A133P, A137E, A137H, A144G, A144H,        A144K, A144L, A144N, A153S, A153V, A176C, A179G, A187G, A187S,        A200G, A216W, A223S, A228S, A230T, A230V, A231V, A232C, A232V,        A272E, A272G, A272K, A272P, A273G, A273L, A273V, A274M, A274R,        D036E, D099A, D099Q, D120E, D181A, D181G, D259A, D259G, D259Q,        D259T, E156A, E156S, E251I, E251L, E251Q, E251T, F058Y, F261C,        F261D, F261K, F261P, G020E, G020F, G020H, G020L, G020N, G020Q,        G020R, G020T, G020Y, G024A, G024P, G053A, G053D, G053E, G053F,        G053L, G053Q, G053S, G053Y, G097K, G097M, G157A, G157S, G160A,        G160L, G166C, G166I, G166Q, G169A, G211K, G215H, G215L, G215S,        G215T, G215W, G258S, H017I, H039S, H226L, H238N, H238Y, I011L,        I011V, I031L, I079F, I079K, I079L, I079M, I079Q, I205A, I205V,        I268L, I268M, K012G, K043F, K043H, K043I, K043N, K043Q, K043T,        K141A, K141R, K141W, K170A, K213A, K213G, K213H, K213I, K213L,        K213N, K213Q, K213R, K213S, K213T, K213V, K237A, K237H, K237I,        K237L, K237N, K237S, K256A, K256G, K256H, K256M, K256P, K256Q,        K256W, K265H, L016E, L042V, L075G, L075H, L075I, L075T, L082A,        L082F, L082H, L082R, L082S, L082T, L090M, L135F, L196M, L209C,        L209H, L209S, L233S, L235M, L235R, L235W, L257C, L257G, L267F,        M050Y, M119C, M119I, M124L, N025C, N025E, N025P, N061A, N061G,        N061I, N061K, N061L, N061Q, N061R, N062S, N062T, N076A, N076P,        N076Q, N076S, N076T, N076V, N078G, N078H, N078K, N078P, N078Q,        N078R, N101F, N117R, N117S, N118D, N118H, N118Q, N118R, N118S,        N118T, N184C, N184E, N184R, N212D, N212R, N212W, N218F, N218G,        N218M, N218P, N218T, N218V, N218W, N240A, N240G, N240Q, N240S,        N240W, N243C, N243G, N243S, N252V, N269H, P005A, P005D, P005M,        P005Q, P014A, P014M, P014R, P014V, P040F, P040R, P040W, P129E,        P129R, P172E, P172K, P194H, P194R, P194W, P201A, P201G, P210L,        P239K, P239R, Q002D, Q002E, Q002G, Q002I, Q002P, Q002V, Q010D,        Q010R, Q019C, Q019D, Q019E, Q019H, Q019L, Q019P, Q019R, Q059A,        Q059E, Q059L, Q059S, Q059T, Q103W, Q185D, Q185K, Q185R, Q185W,        Q206G, Q206H, Q206L, Q206V, Q206W, Q217E, Q217F, Q217H, Q217L,        Q217V, Q245M, Q271A, Q271D, Q271G, Q271L, Q271P, Q271T, Q271Y,        Q275F, Q275L, Q275P, Q275R, S003D, S003F, S063A, S063F, S063G,        S063M, S063R, S063Y, S087C, S087K, S087L, S087M, S087N, S087Y,        S089A, S089D, S089F, S089G, S089H, S089I, S089K, S089R, S089V,        S089Y, S130D, S130E, S130K, S130W, S145G, S145L, S145R, S145T,        S159D, S159L, S159W, S161E, S161R, S162C, S162E, S162W, S163A,        S182E, S182R, S183C, S183D, S183P, S183R, S188D, S188P, S204G,        S204Y, S207G, S224G, S224T, S236C, S236G, S248D, S248H, S248R,        S249E, S249L, S249R, S260A, S260G, S260K, S260Q, S260V, S260Y,        T022L, T055D, T055E, T055I, T055K, T055M, T055S, T055V, T055Y,        T158A, T158G, T158L, T158Q, T158V, T164K, T164Q, T208S, T244D,        T244E, T244R, T253E, T253R, T253Y, T254G, T255D, T255E, T255K,        T255R, V026A, V028I, V028L, V030I, V044C, V044P, V045E, V045G,        V045N, V072L, V081A, V081G, V081H, V081S, V084I, V084M, V095A,        V095C, V143A, V143F, V143H, V143Q, V143T, V143W, V147A, V147Q,        V147S, V148I, V148L, V149C, V149I, V149L, V165L, V180A, V180C,        V180M, V192C, V192F, V192I, V192Q, V192Y, V203A, V203G, V203K,        V203S, V270C, V270L, V270P, W241F, Y006A, Y006M, Y006N, Y006R,        Y006S, Y021C, Y091W, Y104V, Y104W, Y262C, Y262D, Y262E, Y262H,        Y262I, Y262R, Y262V, A088T-A116T, A088T-N109G, A088T-N243V,        A088T-S249A, A116T, A116T-G131H, A116T-N243V, A116T-S162G,        A116T-T158S, A128S-K256R, A128S-L257G, A128S-S248N, A128S-T158S,        G024E-A116T, G024E-K256R, G024E-L257G, G024E-N243V, G024E-T158S,        G131H-S249A, K043Y-A088T, N076D-K256R, N109G, N109G-A116T,        N109G-A128S, N109G-A128S-N243V-K256R, N109G-A128S-N243V-S248A,        N109G-L257G, N109G-N243V, N243V-K256R, N243V-S248N, N243V-S249A,        Q103H-A128S, Q103H-G131H, Q103H-K256R, Q103H-L257G, Q103H-N243V,        Q103H-S248N, Q103H-S249A, Q103H-T158S, S033T-N243V, S033T-T158S,        S063G-A128S, S063G-K256R, S063G-S162G, S063G-T158S, S248N-L257G,        S249A, T158S-S249A, A001E-A128S-G131H-N243V,        A001E-G131H-G169A-N243V, A001E-N109G, A001E-S033T-N109G-N218S,        A088T-A128S, A116T-G169A, A116T-Q206D, A128S-N243V, G024E,        G024E-A088T, G024E-A128S, G024E-G131H, G024E-N218S, G024E-S162G,        G024E-S249A, G169A, G169A-K256R, G169A-L257G, G169A-N218S,        G169A-N243V, G169A-Q206D, G169A-S248N, G169A-S249A, K043Y,        K043Y-A116T, K043Y-G169A, N076D, N109G-G169A, N218S-L257G,        P040E-N109G-A128S-G131H, Q206D-L257G, Q206D-N243V, S033T-A128S,        S033T-A128S-G131H-N243P, S033T-K256R, S033T-L257G, S033T-N076D,        S033T-N109G, S033T-N218S, S033T-P040E-Q103H-N109G,        S033T-Q103H-A128S-G131H, S033T-S063G, S033T-S248N,        S063G-N109G-A128S-G131H, S063G-N243V, S063G-Q206D, S248N-K256R,        T158S-Q206D, A001E-A128S, A001E-G131H, A001E-K256R, A001E-N218S,        A001E-N243V, A001E-S033T, A001E-S063G, A001E-S162G, A088T-K256R,        A088T-N218S, A088T-Q103H, A088T-S162G,        A088T-T158SA116T-A128SA116T-G131HA116T-K256R, A116T-L257G,        A116T-S162G, A116T-S248N, A116T-S249A, A128S-G169A, A128S-N218S,        A128S-S162G, A128S-S249A, G024E, G024E-N109G, G024E-Q103H,        G024E-S033T, G024E-S063G, G024E-S248N, G131H-L257G, G131H-N243V,        G131H-S162G, G131H-T158S, G169A, G169A-L257G, G169A-S248N,        K043Y-A128S, K043Y-G131H, K043Y-K256R, K043Y-L257G, K043Y-N109G,        K043Y-N243V, K043Y-Q103H, K043Y-S063G, K043Y-S162G, K043Y-S248N,        K043Y-S249A, K043Y-T158S, K256R-L257G, N061G-N109G-N243V,        N061S-A128S-N243V-S260P, N061S-N109G-A128S-N243V-S260P,        N061S-N109G-A128S-S260P, N076D-A088T, N076D-A128S, N076D-G169A,        N076D-N218S, N076D-N243V, N076D-S162G, N076D-S248N, N076D-T158S,        N109A-A128S-N243V-K256R, N109G-A128S-G131H-N243V-S248N-K256R,        N109G-A128S-N243V-S248A-K256R,        N109G-A128S-N243V-S248N-K256R-L257G,        N109G-A128S-S162G-N243V-S248N-K256R,        N109G-A128S-T158S-N243V-S248N-K256R, N109G-Q206D, N109G-S162G,        N109G-S249A, N109G-T158S, N109Q-A128S-N243V-K256R,        N109S-A128S-N243V-K256R, N218S-K256R, N218S-N243V,        P040A-N109G-A128S-N243V-S248N-K256R, Q103H, Q103H-A116T,        Q103H-G169A, Q103H-N109G, Q103H-N218S, Q103H-S162G,        S009T-A128S-K141R-N243V, S009T-N109G-A128S-K141R,        S009T-N109G-A128S-K141R-N243V,        S009T-S018T-Y021N-N109G-A128S-K141R, S018T-Y021N-N109G-A128S,        S018T-Y021N-N109G-A128S-N243V,        S018T-Y021N-N109G-A128S-N243V-S248N-K256R, S033T-A088T,        S033T-A116T, S033T-G131H, S033T-K043Y, S033T-L257G, S033T-N109G,        S033T-Q103H, S033T-Q206D, S033T-S162G, S033T-S249A, S063G,        S063G-A088T, S063G-A116T, S063G-L257G, S063G-N076D, S063G-N109G,        S063G-N218S, S063G-Q103H, S063G-S248N, S063G-S249A, S162G-G169A,        S162G-L257G, S162G-N218S, S162G-N243V, S162G-S248N, S162G-S249A,        S248N-S249A, S249A-K256R, S249A-L257G, T158S-G169A, T158S-K256R,        T158S-L257G, T158S-N218S, T158S-S248N, A001E, A001E-A088T,        A001E-A116T, A001E-G169A, A001E-L257G, A001E-N109G,        A001E-S033T-N109G-N243V, A001E-T158S, A088T-G169A, A088T-Q206D,        A116T-N218S, A128S-G131H, A128S-N243V-S248N-K256R, A128S-Q206D,        G024E-K043Y, G024E-N076D, G024E-Q206D, G131H-G169A, G131H-N218S,        G131H-N243V-K256R, G131H-Q206D, G131H-S248N, G169A-K256R,        G169A-N218S, G169A-N243V, G169A-Q206D, K043Y-N076D, K043Y-N218S,        N061P-N109G-G131H-N243V, N061P-N109G-N243V,        N061S-N109G-A128S-N243V-S248N-K256R-S260P, N061S-N109G-N243V,        N076D, N076D-G131H, N076D-L257G, N076D-N109G, N076D-Q103H,        N076D-S249A, N109G-A128S-N243V-S248N,        N109G-A128S-N243V-S248N-K256R, N109G-A128S-S248N-K256R,        N109G-N243P-S248A-K256R, N109G-N243P-S248N-K256R,        N109G-N243V-K256R, N109G-N243V-S248A-K256R, N109G-N243V-S248N,        N109G-N243V-S248N-K256R, N109G-S248N-K256R, N218S-S249A,        N243V-S248N-K256R, Q103H-Q206D, Q206D, Q206D-K256R, Q206D-N218S,        Q206D-S248N, Q206D-S249A,        S009T-N109G-A128S-K141R-N243V-S248N-K256R,        S009T-S018T-Y021N-A128S-K141R-N243V, S018T-Y021N-A128S-N243V,        S018T-Y021N-N061S-A128S-N243V-S260P,        S018T-Y021N-N061S-N109G-A128S-S260P,        S018T-Y021N-S033T-N109G-A128S-N243V-S248N-K256R, S033T,        S033T-G169A, S033T-N076D-A128S-N218S,        S033T-N076D-N109G-A128S-N218S-N243V-S248N-K256R,        S033T-N109G-A128S-N243P-S248N-K256R,        S033T-N109G-A128S-N243V-S248N-K256R, S063G-G131H, S063G-G169A,        S162G-Q206D, T158S-S162G, A088T-A116T-G131H-L257G,        A088T-A116T-G131H-N218S-A274T, A088T-A116T-G131H-N218S-K256R,        A088T-A116T-G131H-N218S-K256R-L257G,        A088T-A116T-G131H-N218S-N243V,        A088T-A116T-G131H-N218S-N243V-L257G,        A088T-A116T-G131H-N218S-N243V-S248N-K256R,        A088T-A116T-G131H-N218S-N243V-S248N-K256R-L257G,        A088T-A116T-G131H-N218S-N243V-S248N-L257G,        A088T-A116T-G131H-N218S-S248N,        A088T-A116T-G131H-N218S-S248N-L257G, A088T-A116T-G131H-N243V,        A088T-A116T-G131H-N243V-K256R, A088T-A116T-G131H-N243V-S248N,        A088T-A116T-G131H-N243V-S248N-A274V,        A088T-A116T-G131H-N243V-S248N-K256R-L257G,        A088T-A116T-G131H-N243V-S248N-K256R-L257G,        A088T-A116T-G131H-N243V-S248N-L257G,        A088T-A116T-G131H-S248N-K256R,        A088T-A116T-G131H-S248N-K256R-L257G,        A088T-A116T-G131H-T158S-K256R,        A088T-A116T-G131H-T158S-K256R-L257G,        A088T-A116T-G131H-T158S-N218S-K256R,        A088T-A116T-G131H-T158S-N218S-K256R-L257G,        A088T-A116T-G131H-T158S-N218S-L257G,        A088T-A116T-G131H-T158S-N218S-N243V-K256R,        A088T-A116T-G131H-T158S-N218S-N243V-K256R-L257G,        A088T-A116T-G131H-T158S-N218S-N243V-L257G,        A088T-A116T-G131H-T158S-N218S-N243V-S248N,        A088T-A116T-G131H-T158S-N218S-N243V-S248N,        A088T-A116T-G131H-T158S-N218S-N243V-S248N-K256R,        A088T-A116T-G131H-T158S-N243V-K256R,        A088T-A116T-G131H-T158S-N243V-K256R,        A088T-A116T-G131H-T158S-N243V-K256R-L257G,        A088T-A116T-G131H-T158S-N243V-L257G,        A088T-A116T-G131H-T158S-N243V-S248N,        A088T-A116T-G131H-T158S-N243V-S248N-K256R-L257G,        A088T-A116T-G131H-T158S-N243V-S248N-K256R-L257G,        A088T-A116T-G131H-T158S-N243V-S248N-L257G,        A088T-A116T-G131H-T158S-S248N-K256R,        A088T-A116T-G131H-T158S-S248N-K256R-L257G, A088T-A116T-K256R,        A088T-A116T-L257G, A088T-A116T-N218S,        A088T-A116T-N218S-N243V-K256R, A088T-A116T-N218S-N243V-N269D,        A088T-A116T-N218S-N243V-S248N,        A088T-A116T-N218S-N243V-S248N-K256R-L257G,        A088T-A116T-N218S-N243V-S248N-L257G, A088T-A116T-N218S-S248N,        A088T-A116T-N218S-S248N-L257G, A088T-A116T-N243V-K256R-L257G,        A088T-A116T-N243V-S248N-K256R, A088T-A116T-S248N,        A088T-A116T-S248N-K256R,        A088T-A116T-T158S-A216S-N218S-N243V-K256R-L257G,        A088T-A116T-T158S-N218S-K256R,        A088T-A116T-T158S-N218S-N243V-K256R,        A088T-A116T-T158S-N218S-N243V-S248N-K256R-L257G,        A088T-A116T-T158S-N218S-S248N, A088T-A116T-T158S-N243V-K256R,        A088T-A116T-T158S-N243V-S248N,        A088T-A116T-T158S-N243V-S248N-K256R,        A088T-A116T-T158S-N243V-S248N-L257G, A088T-A116T-T158S-S248N,        A088T-G131D-T158S-N243V-S248N, A088T-G131H-A138V-N218S-L257G,        A088T-G131H-K256R, A088T-G131H-N218S-N243V-K256R,        A088T-G131H-N218S-N243V-K256R, A088T-G131H-N218S-S248N,        A088T-G131H-N218S-S248N-K256R-L257G, A088T-G131H-N218T-L257G,        A088T-G131H-N243V-L257G, A088T-G131H-N243V-S248N-K256R,        A088T-G131H-S248N, A088T-G131H-S248N-L257G,        A088T-G131H-T158S-K256R, A088T-G131H-T158S-K256R-L257G,        A088T-G131H-T158S-N218S, A088T-G131H-T158S-N218S,        A088T-G131H-T158S-N218S-K256R-L257G,        A088T-G131H-T158S-N218S-N243V-K256R,        A088T-G131H-T158S-N218S-N243V-K256R,        A088T-G131H-T158S-N218S-N243V-S248N,        A088T-G131H-T158S-N218S-S248N,        A088T-G131H-T158S-N218S-S248N-K256R,        A088T-G131H-T158S-N218S-S248N-L257G-I268V,        A088T-G131H-T158S-N243V-K256R,        A088T-G131H-T158S-N243V-K256R-L257G,        A088T-G131H-T158S-N243V-S248N,        A088T-G131H-T158S-N243V-S248N-K256R-L257G,        A088T-G131H-T158S-S248N, A088T-G131H-T158S-S248N-K256R,        A088T-N109G-A116T-G131H-K256R,        A088T-N109G-A116T-G131H-K256R-L257G,        A088T-N109G-A116T-G131H-K256R-L257G,        A088T-N109G-A116T-G131H-N218S-K256R,        A088T-N109G-A116T-G131H-N218S-K256R,        A088T-N109G-A116T-G131H-N218S-K256R-L257G,        A088T-N109G-A116T-G131H-N218S-N243V-L257G,        A088T-N109G-A116T-G131H-N218S-S248N,        A088T-N109G-A116T-G131H-N218S-S248N-L257G,        A088T-N109G-A116T-G131H-N243V-K256R,        A088T-N109G-A116T-G131H-N243V-K256R-L257G,        A088T-N109G-A116T-G131H-N243V-S248N-K256R,        A088T-N109G-A116T-G131H-S248N-K256R-L257G,        A088T-N109G-A116T-G131H-S248N-L257G,        A088T-N109G-A116T-G131H-T158S-K256R,        A088T-N109G-A116T-G131H-T158S-L257G,        A088T-N109G-A116T-G131H-T158S-N218S,        A088T-N109G-A116T-G131H-T158S-N218S-L257G,        A088T-N109G-A116T-G131H-T158S-N218S-N243V,        A088T-N109G-A116T-G131H-T158S-N218S-N243V-K256R,        A088T-N109G-A116T-G131H-T158S-N218S-N243V-L257G,        A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R,        A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,        A088T-N109G-A116T-G131H-T158S-N218S-S248N,        A088T-N109G-A116T-G131H-T158S-N218T-K256R,        A088T-N109G-A116T-G131H-T158S-N243V,        A088T-N109G-A116T-G131H-T158S-N243V-S248N,        A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R-L257G,        A088T-N109G-A116T-G131H-T158S-N243V-S248N-L257G,        A088T-N109G-A116T-G131H-V149A-T158S-N218S-K256R,        A088T-N109G-A116T-K256R, A088T-N109G-A116T-N218S-K256R,        A088T-N109G-A116T-N218S-N243V,        A088T-N109G-A116T-N218S-N243V-K256R-L257G,        A088T-N109G-A116T-N218S-N243V-L257G,        A088T-N109G-A116T-N218S-N243V-S248N-K256R,        A088T-N109G-A116T-N218S-S248N,        A088T-N109G-A116T-N218S-S248N-K256R-L257G,        A088T-N109G-A116T-N243V-K256R,        A088T-N109G-A116T-N243V-S248N-K256R-L257G,        A088T-N109G-A116T-N243V-S248N-L257G, A088T-N109G-A116T-S248N,        A088T-N109G-A116T-S248N-K256R-L257G,        A088T-N109G-A116T-S248N-L257G, A088T-N109G-A116T-T158S,        A088T-N109G-A116T-T158S-K256R, A088T-N109G-A116T-T158S-N218S,        A088T-N109G-A116T-T158S-N218S-L257G,        A088T-N109G-A116T-T158S-N218S-N243V-S248N,        A088T-N109G-A116T-T158S-N218S-N243V-S248N-K256R,        A088T-N109G-A116T-T158S-N218S-N243V-S248N-K256R-L257G,        A088T-N109G-A116T-T158S-N218S-S248N,        A088T-N109G-A116T-T158S-N218S-S248N-K256R,        A088T-N109G-A116T-T158S-N218S-S248N-L257G,        A088T-N109G-A116T-T158S-N243V-K256R,        A088T-N109G-A116T-T158S-N243V-S248N,        A088T-N109G-A116T-T158S-S248N.        A088T-N109G-A116T-T158S-S248N-K256R,        A088T-N109G-A137E-T158S-N218S-N243V-S248N-K256R-L257G,        A088T-N109G-G131H-A152S-T158S-N218S-S248N-K256R,        A088T-N109G-G131H-K256R-L257G, A088T-N109G-G131H-N218S,        A088T-N109G-G131H-N218S, A088T-N109G-G131H-N218S-K256R,        A088T-N109G-G131H-N218S-L257G, A088T-N109G-G131H-N218S-N243V,        A088T-N109G-G131H-N218S-N243V-K256R,        A088T-N109G-G131H-N218S-N243V-K256R-L257G,        A088T-N109G-G131H-N218S-N243V-S248N-K256R,        A088T-N109G-G131H-N218S-S248N,        A088T-N109G-G131H-N218S-S248N-K256R,        A088T-N109G-G131H-N218S-S248N-K256R-L257G,        A088T-N109G-G131H-N243V-K256R,        A088T-N109G-G131H-N243V-K256R-L257G,        A088T-N109G-G131H-N243V-L257G,        A088T-N109G-G131H-N243V-S248N-K256R,        A088T-N109G-G131H-S248N-K256R, A088T-N109G-G131H-S248N-L257G,        A088T-N109G-G131H-T158S, A088T-N109G-G131H-T158S-K256R,        A088T-N109G-G131H-T158S-K256R-L257G,        A088T-N109G-G131H-T158S-N218S-K256R,        A088T-N109G-G131H-T158S-N218S-L257G,        A088T-N109G-G131H-T158S-N218S-N243V,        A088T-N109G-G131H-T158S-N218S-N243V-K256R,        A088T-N109G-G131H-T158S-N218S-N243V-S248N,        A088T-N109G-G131H-T158S-N218S-S248N-L257G,        A088T-N109G-G131H-T158S-N243V-K256R-L257G,        A088T-N109G-G131H-T158S-N243V-S248N,        A088T-N109G-G131H-T158S-N243V-S248N-K256R-L257G,        A088T-N109G-G131H-T158S-N243V-S248N-L257G,        A088T-N109G-G131H-T158S-S248N,        A088T-N109G-G131H-T158S-S248N-L257G,        A088T-N109G-G131H-V149A-K256R-L257G,        A088T-N109G-G154A-N155P-E156T-G157L-T158M-S159E-G160E-S161L,        A088T-N109G-K256R-L257G, A088T-N109G-N218S-K256R,        A088T-N109G-N218S-N243V-L257G,        A088T-N109G-N218S-N243V-S248N-K256R, A088T-N109G-N218S-S248N,        A088T-N109G-N218S-S248N-L257G, A088T-N109G-N243V-S248N-K256R,        A088T-N109G-S248N, A088T-N109G-T158S-N218S,        A088T-N109G-T158S-N218S-K256R-Q271H,        A088T-N109G-T158S-N218S-N243V,        A088T-N109G-T158S-N218S-N243V-K256R-Q275R,        A088T-N109G-T158S-N218S-N243V-S248N-K256R-L257G,        A088T-N109G-T158S-N218S-S248N,        A088T-N109G-T158S-N218S-S248N-K256R,        A088T-N109G-T158S-N218S-S248N-N269D,        A088T-N109G-T158S-N243V-K256R,        A088T-N109G-T158S-N243V-S248N-K256R,        A088T-N109G-T158S-N243V-S248N-K256R-L257G-N269D,        A088T-N109G-T158S-N243V-S248N-L257G,        A088T-N109G-T158S-S248N-K256R-L257G,        A088T-N109G-T158S-S248N-L257G,        A088T-N109G-V147A-N218S-N243V-K256R, A088T-N218S-K256R,        A088T-N218S-L257G-I268V, A088T-N218S-N243V,        A088T-N218S-N243V-K256R, A088T-N218S-N243V-K256R-L257G,        A088T-N218S-N243V-L257G, A088T-N218S-N243V-S248N-K256R-L257G,        A088T-N218S-N243V-S248N-L257G, A088T-N218S-N243V-S248N-N269S,        A088T-N218S-S248N-K256R, A088T-N243V-S248N,        A088T-N243V-S248N-K256R, A088T-N243V-S248N-K256R-L257G,        A088T-N243V-S248N-L257G, A088T-S248N, A088T-S248N-K256R-L257G,        A088T-S248N-L257G, A088T-S248N-L257G-I268V, A088T-T158S-N218S,        A088T-T158S-N218S-K256R, A088T-T158S-N218S-L257G,        A088T-T158S-N218S-N243V-K256R-I268V,        A088T-T158S-N218S-N243V-K256R-L257G,        A088T-T158S-N218S-N243V-S248N-L257G, A088T-T158S-N218S-S248N,        A088T-T158S-N243V-K256R, A088T-T158S-N243V-K256R-L257G,        A088T-T158S-N243V-S248N-K256R, A088T-T158S-N243V-S248N-L257G,        A088T-T158S-S248N, A088T-T158S-S248N-L257G, A088T-V147A-K256R,        A098S-G131H-T158S-N218S-N243V-S248N-K256R-L257G,        A116T-G131H-N218S-K256R, A116T-G131H-N218S-K256R-L257G,        A116T-G131H-N218S-L257G, A116T-G131H-N218S-N243V-S248N-L257G,        A116T-G131H-N218S-S248N-K256R-L257G, A116T-G131H-N243V-S248N,        A116T-G131H-N243V-S248N-L257G,        A116T-G131H-T158S-A231V-N243V-L257G, A116T-G131H-T158S-K256R,        A116T-G131H-T158S-K256R-L257G, A116T-G131H-T158S-N218S-K256R,        A116T-G131H-T158S-N218S-K256R-L257G,        A116T-G131H-T158S-N218S-N243V,        A116T-G131H-T158S-N218S-N243V-K256R,        A116T-G131H-T158S-N218S-N243V-K256R-L257G,        A116T-G131H-T158S-N218S-N243V-L257G,        A116T-G131H-T158S-N218S-N243V-S248N-K256R,        A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,        A116T-G131H-T158S-N218S-N243V-S248N-L257G,        A116T-G131H-T158S-N218S-S248N-K256R,        A116T-G131H-T158S-N218T-L257G, A116T-G131H-T158S-S248N-K256R,        A116T-G131H-T158S-S248N-L257G, A116T-N218S-K256R,        A116T-N218S-K256R-L257G, A116T-N218S-N243V-S248N-K256R,        A116T-N218S-N243V-S248N-K256R-L257G,        A116T-N218S-N243V-S248N-L257G, A116T-N218S-S248N,        A116T-N218S-S248N-K256R, A116T-N218S-S248N-L257G,        A116T-N243V-S248N-L257G, A116T-S248N-L257G,        A116T-T158S-L257G-Q271R, A116T-T158S-N218S-L257G,        A116T-T158S-N218S-N243V-K256R-L257G,        A116T-T158S-N218S-S248N-K256R,        A116T-T158S-N218S-S248N-K256R-L257G,        A116T-T158S-N243V-S248N-K256R,        A116T-T158S-N243V-S248N-K256R-L257G,        G024S-G053S-N078S-G097A-N101S-A128S, G131H-N218S,        G131H-N218S-K256R, G131H-N218S-N243V-K256R,        G131H-N218S-N243V-K256R-L257G, G131H-N218S-N243V-S248N,        G131H-N218S-N243V-S248N-K256R, G131H-N218S-S248N-K256R-L257G,        G131H-N218S-S248N-L257G, G131H-N243V-K256R-L257G,        G131H-N243V-S248N-K256R-L257G, G131H-S248N-K256R,        G131H-T158S-K256R, G131H-T158S-K256R-L257G,        G131H-T158S-N218S-K256R, G131H-T158S-N218S-K256R-L257G,        G131H-T158S-N218S-N243V-K256R-L257G,        G131H-T158S-N218S-N243V-S248N,        G131H-T158S-N218S-S248N-K256R-L257G,        G131H-T158S-N218S-S248N-L257G, G131H-T158S-N243V-K256R,        G131H-T158S-N243V-S248N-K256R, G131H-T158S-S248N-L257G,        G131H-V147I-N218S-S248N-K256R,        I107T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,        I107T-N109G-G131H-N218S-N243V-K256R-L257G, N109G,        N109G-A116T-G131H,        N109G-A116T-G131H-A144V-T158S-S248N-K256R-L257G,        N109G-A116T-G131H-K256R-L257G, N109G-A116T-G131H-L257G,        N109G-A116T-G131H-N218S-N243V-K256R,        N109G-A116T-G131H-N218S-N243V-S248N-K256R,        N109G-A116T-G131H-N243V, N109G-A116T-G131H-N243V-K256R-L257G,        N109G-A116T-G131H-N243V-S248N,        N109G-A116T-G131H-N243V-S248N-K256R,        N109G-A116T-G131H-S248N-K256R, N109G-A116T-G131H-T158S-K256R,        N109G-A116T-G131H-T158S-K256R-L257G,        N109G-A116T-G131H-T158S-L257G, N109G-A116T-G131H-T158S-N218S,        N109G-A116T-G131H-T158S-N218S-K256R-L257G,        N109G-A116T-G131H-T158S-N218S-L257G,        N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,        N109G-A116T-G131H-T158S-N218S-S248N-K256R,        N109G-A116T-G131H-T158S-N218S-S248N-K256R-L257G,        N109G-A116T-G131H-T158S-N243V-L257G,        N109G-A116T-G131H-T158S-S248N-L257G,        N109G-A116T-G131H-V147A-T158S-N218S-K256R-L257G,        N109G-A116T-G131H-V149A-T158S-N218S-N243V-S248N-L257G,        N109G-A116T-K256R, N109G-A116T-N218S-K256R,        N109G-A116T-N218S-N243V, N109G-A116T-N218S-N243V-L257G,        N109G-A116T-N218S-N243V-S248N-I268V,        N109G-A116T-N218S-N243V-S248N-K256R-L257G,        N109G-A116T-N218S-N243V-S248N-L257G, N109G-A116T-N243V-S248N,        N109G-A116T-N243V-S248N-K256R, N109G-A116T-N243V-S248N-L257G,        N109G-A116T-S248N-K256R, N109G-A116T-T158S,        N109G-A116T-T158S-N218S-N243V-S248N-K256R,        N109G-A116T-T158S-N218S-N243V-S248N-K256R-L257G,        N109G-A116T-T158S-N218S-S248N-K256R,        N109G-A116T-T158S-N243V-L257G, N109G-A116T-T158S-N243V-S248N,        N109G-A116T-T158S-S248N, N109G-A116T-T158S-S248N-K256R,        N109G-G131H, N109G-G131H-K256R, N109G-G131H-N218S-K256R,        N109G-G131H-N218S-K256R-L257G, N109G-G131H-N218S-N243V-L257G,        N109G-G131H-N218S-N243V-S248N-K256R,        N109G-G131H-N218S-N243V-S248N-K256R-L257G,        N109G-G131H-N218S-N243V-S248N-L257G,        N109G-G131H-N218S-S248N-L257G, N109G-G131H-N243V,        N109G-G131H-N243V-K256R, N109G-G131H-N243V-S248N,        N109G-G131H-N243V-S248N-K256R-L257G,        N109G-G131H-N243V-S248N-L257G,        N109G-G131H-T158S-N218S-K256R-L257G,        N109G-G131H-T158S-N218S-L257G, N109G-G131H-T158S-N218S-N243V,        N109G-G131H-T158S-N218S-N243V-K256R-L257G,        N109G-G131H-T158S-N218S-N243V-S248N,        N109G-G131H-T158S-N218S-N243V-S248N-K256R-L257G,        N109G-G131H-T158S-N218S-N243V-S248N-L257G,        N109G-G131H-T158S-N218S-S248N-K256R-L257G,        N109G-G131H-T158S-N243V-K256R-I268V,        N109G-G131H-T158S-N243V-S248N,        N109G-G131H-T158S-N243V-S248N-K256R,        N109G-G131H-T158S-N243V-S248N-L257G, N109G-G131H-T158S-S248N,        N109G-G131H-T158S-S248N-K256R-L257G,        N109G-K141E-N218S-S248N-L257G, N109G-N218S,        N109G-N218S-N243V-K256R, N109G-N218S-N243V-L257G,        N109G-N218S-N243V-S248N-S260F, N109G-N218S-S248N,        N109G-N218S-S248N-K256R, N109G-N243V-S248N-L257G,        N109G-N243V-S248N-L257G-Q275R,        N109G-S182F-S204F-S207L-N218S-S236F-S248N-L257G,        N109G-T158S-K256R-L257G, N109G-T158S-L257G,        N109G-T158S-N218S-N243V-K256R, N109G-T158S-N218S-N243V-S248N,        N109G-T158S-N218S-N243V-S248N-L257G, N109G-T158S-N243V-K256R,        N109G-T158S-N243V-S248N-K256R, N109G-T158S-N243V-S248N-L257G,        N109G-T158S-S248N-L257G, N218S-N243V-L257G,        N218S-N243V-S248N-K256R, N243V-K256R-L257G,        N243V-S248N-L257G-Q271R,        P057Q-A088T-N109G-A116T-G131H-T158S-N218S-S248N,        S003P-A116T-N218S-K256R,        S003P-N109G-G131H-N218S-N243V-S248N-K256R-L257G,        S248N-K256R-L257G, T158S-K256R-L257G, T158S-N218S-A272V,        T158S-N218S-K256R-L257G, T158S-N218S-L233S, T158S-N218S-N243V,        T158S-N218S-N243V-K256R-L257G, T158S-N218S-N243V-L257G,        T158S-N218S-N243V-S248N-K256R, T158S-N218S-S248N-K256R,        T158S-N243V, T158S-N243V-K256R-L257G, T158S-N243V-S248N,        T158S-N243V-S248N-K256R-N269D,        V004A-N109G-A116T-T158S-N218S-S248N-L257G,        A088T-A098S-N218S-K256R, A088T-A116T-G131H-K256R,        A088T-A116T-G131H-K256R-L257G-L267M,        A088T-A116T-G131H-N218S-N243V-K256R,        A088T-A116T-G131H-N218S-N243V-K256R-L257G,        A088T-A116T-G131H-N218S-N243V-S248N,        A088T-A116T-G131H-N218S-S248N,        A088T-A116T-G131H-N218S-S248N-K256R,        A088T-A116T-G131H-N218S-S248N-K256R, A088T-A116T-G131H-N243V,        A088T-A116T-G131H-S248N, A088T-A116T-G131H-S248N-L257G,        A088T-A116T-G131H-S248N-L257G, A088T-A116T-G131H-T158S-N218S,        A088T-A116T-G131H-T158S-N218S-K256R-L257G,        A088T-A116T-G131H-T158S-N218S-N243V-S248N-K256R,        A088T-A116T-G131H-T158S-N218S-N243V-S248N-L257G,        A088T-A116T-G131H-T158S-N218S-S248N,        A088T-A116T-G131H-T158S-N218S-S248N-K256R,        A088T-A116T-K256R-L257G, A088T-A116T-N218S-I268V,        A088T-A116T-N218S-K256R, A088T-A116T-N218S-N243V-Q271R,        A088T-A116T-N218S-N243V-S248N-K256R,        A088T-A116T-N218S-N243V-S248N-K256R-Q275R,        A088T-A116T-N218S-S248N, A088T-A116T-N218S-S248N-K256R,        A088T-A116T-N243V-S248N-K256R, A088T-A116T-T158S,        A088T-A116T-T158S-N218S-K256R,        A088T-A116T-T158S-N218S-N243V-L257G,        A088T-A116T-T158S-N218S-N243V-S248N-L257G,        A088T-A116T-T158S-N218S-S248N,        A088T-A116T-T158S-N218S-S248N-K256R,        A088T-A116T-T158S-N218S-S248N-K256R-L257G,        A088T-A116T-T158S-N218S-S248N-L257G,        A088T-A116T-T158S-S248N-L257G, A088T-G131H, A088T-G131H,        A088T-G131H-N218S-K237R-K256R-L257G, A088T-G131H-N218S-K256R,        A088T-G131H-N218S-K256R-L257G,        A088T-G131H-N218S-N243V-K256R-L257G,        A088T-G131H-N218S-N243V-L257G, A088T-G131H-N218S-N243V-L257G,        A088T-G131H-N218S-N243V-S248N,        A088T-G131H-N218S-N243V-S248N-K256R,        A088T-G131H-N218S-N243V-S248N-K256R-L257G,        A088T-G131H-N218S-N243V-S248N-K256R-L257G,        A088T-G131H-N218S-S248N, A088T-G131H-N243V,        A088T-G131H-N243V-K256R, A088T-G131H-N243V-K256R-L257G,        A088T-G131H-N243V-S248N, A088T-G131H-N243V-S248N,        A088T-G131H-N243V-S248N-K256R, A088T-G131H-S248N,        A088T-G131H-S248N-K256R, A088T-G131H-T158S-N218S-K256R-L257G,        A088T-G131H-T158S-N218S-L257G, A088T-G131H-T158S-N218S-N243V,        A088T-G131H-T158S-N218S-N243V-K256R-L257G,        A088T-G131H-T158S-N218S-N243V-S248N-K256R,        A088T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,        A088T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,        A088T-G131H-T158S-N218S-N243V-S248N-L257G,        A088T-G131H-T158S-N218S-S248N-K256R,        A088T-G131H-T158S-N218S-S248N-K256R-L257G,        A088T-G131H-T158S-S248N-K256R-L257G, A088T-L257G,        A088T-N109G-A116T-G131H-N218S-L257G,        A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R,        A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R-L257G,        A088T-N109G-A116T-G131H-N218S-N243V-S248N-N269D,        A088T-N109G-A116T-G131H-N218S-N243V-S248N-Q275R,        A088T-N109G-A116T-G131H-N218S-S248N-K256R-L257G,        A088T-N109G-A116T-G131H-N243V-S248N,        A088T-N109G-A116T-G131H-T158S,        A088T-N109G-A116T-G131H-T158S-N218S-L257G-I268V,        A088T-N109G-A116T-G131H-T158S-N218S-N243V-K256R-L257G,        A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N,        A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-L257G,        A088T-N109G-A116T-G131H-T158S-N218S-S248N-L257G,        A088T-N109G-A116T-G131H-T158S-S248N,        A088T-N109G-A116T-G131H-W241L-S248N-K256R-L257G,        A088T-N109G-A116T-K256R, A088T-N109G-A116T-N218S-K256R-L257G,        A088T-N109G-A116T-T158S-N218S-K256R-L257G,        A088T-N109G-A116T-T158S-N218S-N243V-S248N-L257G,        A088T-N109G-A116T-T158S-N243V-S248N-K256R,        A088T-N109G-G131H-A138V-T158S-N218S-N243V-S248N-L257G,        A088T-N109G-G131H-N218S-N243V-S248N-L257G,        A088T-N109G-G131H-N218S-S248N-K256R-L257G-Q275R,        A088T-N109G-G131H-N243V-S248N,        A088T-N109G-G131H-N243V-S248N-K256R-L257G,        A088T-N109G-G131H-T158S-L233S-N243V-S248N,        A088T-N109G-G131H-T158S-N218S-N243V-K256R-L257G,        A088T-N109G-G131H-T158S-N218S-N243V-S248N-K256R-L257G,        A088T-N109G-G131H-T158S-N218S-N243V-S248N-L257G,        A088T-N109G-G131H-T158S-N218S-S248N-K256R,        A088T-N109G-G131H-T158S-N243V-S248N-K256R,        A088T-N109G-G131H-T158S-S248N-K256R,        A088T-N109G-G131H-T158S-S248N-K256R-L257G,        A088T-N109G-N218S-K256R-L257G, A088T-N109G-N218S-N243V-K256R,        A088T-N109G-N218S-N243V-S248N,        A088T-N109G-N218S-N243V-S248N-K256R-L257G,        A088T-N109G-N218S-S248N-K256R, A088T-N109G-N243V-K256R,        A088T-N109G-S248N-K256R-L257G, A088T-N109G-T158S,        A088T-N109G-T158S-K256R-L257G,        A088T-N109G-T158S-N218S-N243V-K256R,        A088T-N109G-T158S-N218S-N243V-K256R-L257G,        A088T-N109G-T158S-N218S-N243V-S248N-K256R,        A088T-N109G-T158S-N218S-N243V-S248N-L257G,        A088T-N109G-T158S-S248N, A088T-N218S-N243V-S248N,        A088T-N218S-N243V-S248N-K256R, A088T-N218S-N243V-S248N-K256R,        A088T-N218S-S248N, A088T-N218S-S248N-L257G,        A088T-S248N-K256R-L257G, A088T-T158S-K256R,        A088T-T158S-N218S-N243V-K256R, A088T-T158S-N218S-N243V-L257G,        A088T-T158S-N218S-N243V-S248N,        A088T-T158S-N218S-N243V-S248N-K256R,        A088T-T158S-N218S-N243V-S248N-K256R-L257G,        A088T-T158S-N218S-S248N-K256R, A088T-T158S-N218S-S248N-L257G,        A088T-T158S-N218S-S248N-L257G-Q275K, A088T-T158S-N243V,        A088T-T158S-N243V-S248N, A088T-T158S-S248N-K256R-L257G,        A088T-V147I-N218S-N243V-K256R-L257G, A116T-G131H-L257G,        A116T-G131H-N218S-N243V, A116T-G131H-N218S-N243V-K256R-L257G,        A116T-G131H-N218S-N243V-S248N-K256R-L257G,        A116T-G131H-N218S-S248N, A116T-G131H-N218S-S248N-K256R,        A116T-G131H-N243V-S248N-K256R,        A116T-G131H-T158S-N218S-N243V-S248N,        A116T-G131H-T158S-N218S-S248N, A116T-G131H-T158S-N243V-K256R,        A116T-G131H-V130I-N218S-N243V-S248N,        A116T-K141E-N218S-N243V-S248N-K256R-L257G,        A116T-N218S-N243V-S248N, A116T-N218T-N243V-S248N,        A116T-N243V-K256R-L257G, A116T-S248N-K256R,        A116T-T158S-N218S-K256R, A116T-T158S-N218S-K256R-L257G,        A116T-T158S-N218S-N243V-L257G, A116T-T158S-N218S-N243V-S248N,        A116T-T158S-N218S-S248N-L257G, G024S-G053S-N078S-G097A-N101S,        G053S-A088T-N109G-A116T-G131H-T158S-G169S-N218S-S248N-K256R-L257G,        G131H-K141R-T158S-N218S-K256R, G131H-K256R,        G131H-N218S-K256R-L257G, G131H-N218S-N243V-S248N-L257G,        G131H-N218S-S248N-K256R, G131H-N243V-S248N,        G131H-N243V-S248N-L257G, G131H-T158S-N218S,        G131H-T158S-N218S-N240H-N243V-S248N-K256R-L257G,        G131H-T158S-N218S-N243V,        G131H-T158S-N218S-S248N-K256R-L257G-N269S,        G131H-T158S-N243V-L257G, G131H-T158S-N243V-S248N,        G131H-T158S-S248N, K256R, N109G-A116T-G131H-N218S-N243V,        N109G-A116T-G131H-N218S-N243V-L257G,        N109G-A116T-G131H-N218S-S248N-L257G,        N109G-A116T-G131H-N218S-W241R-N243V-K256R,        N109G-A116T-G131H-S248N-L257G,        N109G-A116T-G131H-T158S-N218S-K256R,        N109G-A116T-G131H-T158S-N218S-N243V-K256R-L257G,        N109G-A116T-G131H-T158S-N218S-N243V-S248N,        N109G-A116T-G131H-T158S-N218S-N243V-S248N-L257G,        N109G-A116T-G131H-T158S-N243V-K256R-L257G,        N109G-A116T-G131H-T158S-N243V-S248N-K256R,        N109G-A116T-G131H-T158S-S248N-K256R-L257G,        N109G-A116T-I234T-N243V-S248N-K256R-L257G,        N109G-A116T-N218S-N243V-S248N, N109G-A116T-N243V-K256R-L257G,        N109G-A116T-T158S-N218S-K237R-N243V-S248N,        N109G-G131H-N218S-N243V-S248N, N109G-G131H-S248N,        N109G-G131H-T158S, N109G-G131H-T158S-L257G,        N109G-G131H-T158S-N218S-N243V-S248N-K256R,        N109G-G131H-T158S-N218S-S248N-K256R,        N109G-G131H-T158S-N218S-S248N-L257G,        N109G-G131H-T158S-N243V-S248N-K256R-L257G,        N109G-G131H-T158S-S248N-K256R, N109G-N218S-K256R-L257G,        N109G-N218S-N243V-S248N-K256R, N109G-N218S-S248N-L257G,        N109G-S248N, N109G-T158S-N218S, N109G-T158S-N218S-N243V,        N109G-T158S-N243V-L257G, N218S-N243V-K256R, N218S-N243V-S248N,        N218S-S248N, N218S-S248N-K256R, N243V-L257G,        S003P-N109G-A116T-G131H-N218S-N243V-S248N,        S003P-N109G-A116T-G131H-T158S-N218S-K256R,        S105H-W106G-I107L-I108S-N109A-G110A-I111S-E112N-W113G-A114P,        S248N-L257G, T158S-N218S-K256R, T158S-N218S-L233S-S248N,        T158S-N218S-L257G, T158S-N218S-N243V-K256R,        T158S-N218S-N243V-S248N, T158S-N218S-N243V-S248N-L257G,        T158S-N218S-S248N-K256R-L257G, T158S-N218S-S248N-L257G,        T158S-N243V-S248N-L257G,        V004L-A088T-G131H-T158S-N218S-S248N-L257G, A133V-S260N,        N061S-S260P, P014T-S037T, S009T-K141F, S009T-K141R, S018F-S162L,        S018L-Y021S, S018P-D120N, S018T-S162P, S018T-Y021N, S018Y-K213R,        S161P-S162L, S161P-S260P, T253A-S260P, A134T-S260G, I115V-N184Y,        N025K-S037P, Q010L-S037P, Q019L-S260N, Q019L-S260P, S037P-T254S,        S161P-T253A, N061S-S260P, Q010L-S037P, S009T-K141F, S018L-Y021S,        S018T-S162P, S018T-Y021N, S018Y-K213R, S037P-T254S, S161P-S260P,        S161P-T253A, T253A-S260P, A133V-S260N, A134T-S260G, I115T-S183T,        I115V-N184Y, N061D-S260I, Q019L-S260N, Q019L-S260P, S183T-S249R,        N61D-Q206R, Y262N-Q275R, K43R-N76S, K170R-D259G, Y6F-S249C,        Q19A-N109S, H17L-Q19A, Q19R-Q185R, S18Y-V203A, S161E-S260T,        S18K-V203I, V4A-T55A, N252S-L257H, S249R-Y262H, N61L-Q206H,        N184Y-Y262N, Q19R-N25D, A74S-P129Q, K27R-D120H, V4A-T55A,        Y21H-N252H, K27R-N269D, A98T-T158A, I79V-Q217H, S9L-N218S,        V4A-Y6F, V203A-Q217R, T22S-T242S, N76P-N212S, S37T-S260P,        T55A-V147A, V4A-Y6F, Y262N-Q275R, G160R-T244A, N25D-Q185R,        G211V-T244A, S9L-N218S, A144H-T244A, Y21H-N252H, A1Y-Q275R,        V198L-D259G, K141I-S248N, S183T-R186K, S161E-Q185H, P129S-K136R,        K43N-S163T, S37G-Q275H, Y6F-S249C, N62Y-T244A, S260P-Q275R,        S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V,        S33T-N61G-S63G-N109G-A128S-N218S-N243V,        S33T-S63G-N109G-A128S-N218S-N243V,        S33T-T55P-N61P-S63G-N109Q-A128S-G131H-S224A-N243V,        N61P-S63G-N109Q-A128S-G131H-G169A-S224A-N243V-S249Q,        S33T-N61G-A88T-N109G-A116T-A128S-N218S-N243V,        S33T-N109G-A128S-N218S-N243V, S33T-N76D-N109G-A128S-N218S-N243V,        S33T-N76D-N109G-A128S-N218S-N243V-S248N-K256R,        S33T-N61G-N109G-A128S-N218S-N243V, S33T-A128S-N218S,        A1G-N61P-S63G-N109Q-A128S-G131H-S224A-N243V,        N61P-S63G-N109Q-A128S-G131H-S224A-N243V-S249Q,        N61P-S63G-N109Q-A128S-G131H-S224A-N243V,        S63G-N109Q-A128S-G131H-S224A-N243V, N109G-A114S-A128S,        N109G-A114S-A128S-S183L-S224A, N109G-A114S-A128S-S224A,        N109G-A114S-A128S-S224A-N243V,        A88T-N109G-A116T-A128S-S224A-N243V,        N61G-A88T-N109G-A116T-A128S-S224A-N243V,        N109G-A114S-A128S-N243V, N109G-A128S-S224A-N243V,        N109G-A128S-S224A, N109G-A128S-S183L-S224A,        N61G-N109G-A128S-S224A, N109G-A128S-S183L, S33T-N76D,        N109S-A128S-S224A, N101Q-N109Q-A128S-P129S-S130T-S224A-N243V,        S63G-N109Q-A128S-S224A-N243V, N109M-A128S-S224A-N243V,        S63G-N109G, N109G-K256R, S63G-N76D,        S33T-N109G-A128S-G169A-N218S-N243V,        S33T-N109G-A128S-N218S-S224A-N243V, N062L, N062L-S063G,        N062S-S063G-Q217L, N062S-S063L-Q217L, N062S-S063N, N062S-S063R,        S063G, S063G-Q217L, S063G-Q217L-M222S, S063L-Q217L, S063N,        S063N-Q217L, D099N-K141Y-K213Q, D099N-K141Y-K256Q,        K043T-K141Y-E156Q, N062L-Q217E, N062L-Q217L, N062L-S063G-Q217E,        N062L-S063L, N062L-S063N-Q217L, N062S-Q217L, N062S-S063G,        N062S-S063L, N062S-S063N-Q217L, N062S-S063R-Q217E, S063G-Q217E,        S063N-Q217E, S063R, S063R-Q217E and S063R-Q217L;-   e) a protease, said protease being selected from the group    consisting of    -   (i) a variant of a parent protease, said parent protease having        at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98%        or 99% or even 100% identity to SEQ ID NO:4, said variant        comprising a group of mutations selected from the following        groups of mutations: Q059V-S078N-I108V-V147Q-G211A-N252Q,        S078N-I108V-V147Q-G211A, S078N-S087E-M124I-S224A,        S078N-I108V-V147Q-N252Q, S078N-I108V-V147Q-G211A-N252Q, and        Q059V-S078N-I108V-V147Q-G211A-N252Q;    -   (ii) a variant of a parent protease, said parent protease,        having at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97%        or 98% or 99% or even 100% identity to SEQ ID NO:2, said variant        comprising a group of mutations selected from the following        groups of mutations: G097A-M124V-Y167A-Y217Q, V068A-Y167A-Y217Q,        G097A-I111V-M124V-Y167A, I111V-M124V-Y167A-Y217Q,        V068A-I111V-Y167A-Y217Q, G097A-I111V-M124V-Y167A-Y217Q,        P052L-V068A-I111V, G097A-N123A-Y217Q,        N061P-N062Q-G097A-G100D-Y217Q, N061P-S101N-G102A-G128S-Y217Q,        Y217Q, N061P-G102A-G128S-Y217Q, N061P-S101N-G102A-G128S-Y217Q,        S078N-G097A-I111V-N123Q-Y217Q, G102A-N123Q-Y217Q,        N061P-G102A-G128S-Y217Q, S078N-G097A-I111V-N123Q-Y217Q, and        G102A-N123Q-Y217Q; and    -   (iii) a variant of a parent protease, said parent protease        having at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97%        or 98% or 99% or even 100% identity to SEQ ID NO:2, said variant        comprising three, four, five or six of the following mutations:        X24G/R, X53G, X78N, X101N, X128A/S and X217L/Q, said variant        optionally comprising at least one group of mutations selected        from the following groups of mutations: A001D, A001H, A001N,        A015C, A048C, A048E, A085T, A133R, A137R, A142C, A144D, A144R,        A152S, A153G, A187P, A187Q, A187T, A187V, A216R, A230S, A272R,        A273H, A273T, A274H, D036N, D036S, D181H, D181T, D259N, D259P,        D259S, E156G, E156H, E156L, E156Q, E156V, E251C, F189S, F189T,        F189W, F189Y, F261E, G020C, G024D, G053M, G053R, G097R, G131D,        G131R, G157N, G160R, G160V, G166L, G166W, G211E, G215D, G258A,        G258D, G258P, I011T, I031C, I079E, I079R, I175L, I205C, K012H,        K012N, K027A, K027N, K027S, K043A, K043D, K043E, K043G, K043L,        K043M, K043P, K043V, K043W, K043Y, K136H, K141H, K141L, K141M,        K141N, K141Q, K141T, K141V, K170G, K170S, K237T, K237V, K256D,        K256S, K256T, K256V, K265N, K265S, L042F, L042M, L082E, L209A,        L209E, L209G, L209R, L233G, L235V, L257D, L257E, L257P, L257R,        L257W, L267E, M050L, N056D, N056S, N061C, N061D, N062A, N062H,        N062L, N062V, N062Y, N076D, N076L, N076M, N078D, N078F, N101D,        N101R, N118A, N212C, N212E, N218C, N218D, N218E, N252D, N252E,        P014F, P014K, P057A, P057W, P172R, P194E, P201T, P210E, Q059C,        Q059D, Q059R, Q185E, Q206D, Q217A, Q217K, Q217R, Q245A, Q245D,        Q245E, Q245H, Q245R, Q271E, Q271F, Q271R, Q271W, Q275G,        Q275IR186I, R186L, R186V, R186W, S003E, S009C, S009E, S018C,        S037D, S037E, S037H, S037R, S037Y, S038D, S038P, S038R, S038Y,        S063L, S087D, S087R, S089C, S089E, S130C, S130R, S145D, S159C,        S159P, S161C, S173T, S182C, S188E, S188F, S188K, S188L, S188R,        S188W, S190A, S190G, S190T, S204R, S236D, S236E, S248C, S248E,        S260C, S260E, S260R, T022P, T055C, T055W, T071A, T158D, T158E,        T158P, T158R, T158Y, T164R, T242D, T242G, T255C, V004E, V004T,        V045C, V045D, V045R, V045T, V051H, V081R, V143C, V143E, V143G,        V192G, V203C, V203D, V203E, V203M, V203R, V270G, W241L, Y214H,        Y214Q, A001E, A133E, A187L, A187N, A216C, A216H, A273Q, D099H,        D259H, E156C, E195G, F189H, G131C, G146A, G166V, G215C, G215E,        I107L, K012A, K012S, K012T, K043C, K170C, K256C, K256E, K265G,        K265Y, L233EM222F, M222S, N062Q, N076E, N078E, N184P, N218R,        P005V, P014D, Q002K, Q002L, Q002R, Q010W, Q271C, R186H, S049C,        S063C, S063D, S105T, S188C, S190C, S204E, T055R, T164G, V004D,        V044T, V045I, V165C, V180S, Y006C, Y006D, Y006E, Y104T, A001C,        A187C, A230G, A273D, A273P, D036Q, F189G, F189L, F189R, G157T,        G178A, I031F, I111M, K012F, K012L, K027T, K043R, K136G, K141G,        K170Q, M222A, M222L, N062R, N117G, N269C, P005W, P129V, P239A,        P239H, P239T, Q059W, Q217G, Q275A, R186A, S191G, T164A, T220A,        A001P, A187F, A187W, A273R, D041C, D060G, D197T, F189A, G046D,        G157P, K012C, K012E, K012W, L042C, M222T, N062C, P239G, P239N,        Q217C, R186M, S049T, S089P, S125A, S173V, V044A, A001E,        A001E-A128S, A001E-G024E, A001E-G131H, A001E-G169A, A001E-L257G,        A001E-218S, A001E-Q103H, A001E-S063G, A001E-S248N, A001E-S249A,        G024E-N076D, K043Y-N076D, K043Y-Q206D, N076D-A116T, N076D-G169A,        N076D-Q206D, Q103H-Q206D, S033T-G169A,        S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243P,        S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243V, A001E-K043Y,        A001E-N076D, A001E-N076D-N109G-A128S, A001E-Q206D, G024E-Q206D,        A001E-A128S-G131H-N243V, A001E-G131H-G169A-N243V,        A001E-S033T-N109G-N218S, A116T-G169A, A116T-Q206D, G169A-S249A,        K043Y-G169A, N109G-G169A, P040E-N109G-I28S-G131H, Q206D-L257G,        S033T-A128S-G131H-N243P, S033T-A128S-G131H-N243V,        S033T-P040E-Q103H-N109G, S033T-Q103H-A128S-G131H,        S063G-N109G-A128S-G131H, S063G-Q206D, T158S-Q206D,        A001E-N076D-N109G-A128S,        A015S-A088T-N109G-G131H-T158S-N218S-S248N,        A088T-A098S-G131H-S248N-K256R-L257G,        A088T-A116T-G131H-N218S-N243V-K256R-L257G,        A088T-A116T-G131H-T158S-L257G,        A088T-A116T-G131H-T158S-N218S-L257G,        A088T-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,        A088T-A116T-G131H-T158S-N218S-N243V-S248N-L257G,        A088T-A116T-N218S-L257G, A088T-A116T-T158S-K256R,        A088T-A116T-T158S-S248N-K256R-L257G,        A088T-G131H-K141E-N218S-N243V-S248N-L257G, A088T-G131H-K256R,        A088T-G131H-N218S-K256R, A088T-G131H-N218S-N243V-S248N-K256R,        A088T-G131H-N218S-N243V-S248N-L257G,        A088T-G131H-N218S-S248N-K256R, A088T-G131H-T158S-S248N-K256R,        A088T-G131H-T158S-S248N-K256R-L257G,        A088T-I107T-N109G-G131H-N218S-S248N-K256R,        A088T-N109G-A116T-G131H-D140G-T158S-N218S-N243V-K256R,        A088T-N109G-A116T-G131H-N218S-N243V-K256R,        A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R,        A088T-N109G-A116T-G131H-T158S-N218S-S248N-L257G,        A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R-I268V,        A088T-N109G-A116T-G131H-V149A-N218S-S248N-K256R-L257G,        A088T-N109G-A116T-N218S-N243V-S248N-K256R-L257G,        A088T-N109G-A116T-T158S-K256R-L257G,        A088T-N109G-A116T-T158S-N218S-N243V-L257G,        A088T-N109G-D140G-N243V,        A088T-N109G-G131H-D140G-T158S-N243V-S248N-K256R,        A088T-N109G-G131H-K141E-T158S-N218S-K256R,        A088T-N109G-G131H-N218S-S248N,        A088T-N109G-G131H-N218S-S248N-K256R-Q271R,        A088T-N109G-G131H-N218S-S248N-L257G,        A088T-N109G-G131H-T158S-K256R,        A088T-N109G-G131H-T158S-N218S-S248N-K256R,        A088T-N109G-G131H-V149L-T158S-K256R-L257G,        A088T-N109G-T158S-N218S,        A088T-N109G-T158S-N218S-K256R-L257G-Q271K,        A088T-N109G-T158S-N218S-L257G, A088T-N109G-T158S-S248N-K256R,        A088T-N218S-S248N-L257G-Q27R, A088T-T158S-N218S-K256R-L257G,        A088T-T158S-N218S-N243V-K256R,        A088T-Y104H-A116T-G131H-N218S-N243V,        A116T-G131H-K141E-N218S-N243V-S248N-L257G,        A116T-G131H-N218S-N243V-S248N-K256R,        A116T-G131H-T158S-N218S-S248N-L257G-N269D,        A116T-G131H-T158S-N218S-S248N-Q271R,        A116T-G131H-T158S-N243V-S248N,        A116T-G157E-T158S-N243V-S248N-K256R, A116T-T158S-N218S,        G131H-N218S-L257G, G131H-N218S-S248N,        G131H-T158S-N218S-N243V-S248N-K256R-L257G,        G131H-T158S-N218S-N243V-S248N-L257G,        G131H-T158S-N218S-S248N-I268V, I107T-N109G-G131H-N218S-L257G,        L090I-N109G-T158S-N243V, L257G,        N109G-A116T-G131H-T158S-N218S-K256R-L257G-Q271R,        N109G-A116T-N218S-W241R-N243V-S248N-K256R-L257G,        N109G-G131H-K141E-L257G, N109G-G131H-N218S-N243V,        N109G-T158S-N218S-N243V-L257G, N109G-T158S-N218S-S248N-K256R,        N109G-T158S-N243V-S248N-K256R-L257G, N218S-S248N-K256R-L257G,        S003P-N109G-G131H-T158S-L257G, S003P-S248N-L257G,        T158S-S248N-K256R-L257G,        V004A-A088T-G131H-N218S-N243V-S248N-L257G,        Y006H-N218S-N243V-S248N, Y104H-N109G-G131H-N243V-S248N,        A088T-A116T-T158S-N218S-N243V-S248N-K256R,        A088T-A116T-T158S-N243V,        A088T-G131H-T158S-N218S-I234T-S248N-L257G,        A088T-G131H-T158S-N218S-N243V-S248N-K256R,        A088T-G131H-V149L-T158S-N243V-S248N-K256R-L257G,        A088T-I107T-N109G-G131H-N218S-A223G-S248N-K256R,        A088T-K213N-N243V-S248N-K256R, A088T-K256R-L257G,        A088T-N109G-A116T-G131H-A232S-N243V-K256R,        A088T-N109G-A116T-G131H-D140G-S248N-L257G,        A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R-L257G,        A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N,        A088T-N109G-A116T-G131H-T158S-N243V-S248N-L257G,        A088T-N109G-A116T-M124I-G131H-T158S-N218S-S248N-L257G,        A088T-N109G-A116T-V148A-N218S-N243V,        A088T-N109G-G131H-N218S-N243V-S248N,        A088T-N109G-N218S-S248N-T255K-K256R-L257G,        A088T-T158S-N218S-L257G, A088T-T158S-N218S-Q245K-S248N-K256R,        A088T-T158S-N218S-S248N-K256R, A116T-G131H-N218S-N243V-K256R,        A116T-G131H-N218S-W241R-N243V-S248N-K256R-L257G,        A116T-G131H-T158S-N218S-L257G,        A116T-G131H-V150A-T158S-N243V-S248N-K256R-L257G,        I107T-G131H-T158S-N243V-S248N-K256R-L257G,        N109G-A116T-K141E-T158S-N218S-N243V-L257G,        N109G-A116T-T158S-N218S-N243V-S248N, T158S-N243V-S248N-K256R,        T158S-N243V-S248N-K256R-L257G, A088T-A116T-G131H-G146C,        A088T-A116T-N218S, A088T-A116T-T158S-N243V-K256R-L257G,        A088T-A138E-N218S-N243V-K256R,        A088T-N109G-A116T-G131H-T158S-N218S-N243F-S248N,        A088T-T158S-V203I-N218S-K256R-L257G,        A116T-D140G-T158S-N218S-N243V-S248N,        A088T-A116T-T158S-K256R-L257G,        A088T-A116T-T158S-N218S-N243V-S248N-E251K-K256R-L257G,        A088T-I108T-N109G-G131H-T158S-N218S-S248N-K256R-L257G,        A088T-N109G-A116T-G131H-K141E-N218S,        A088T-N109G-W241R-S248N-K256R, G065D-A088T-G131H-N243V-S248N,        A045S-S236G, G024A-S037W, I031V-S038W, N061D-S260I, Q010R-S037T,        I115T-S183T, N025K-P129K, N025K-P129R, A045S-S236Y, S162L-D181H,        I031V-S038W, N025K-S037P, N025K-P129R, Y21H-D259G, A133V-D259N,        I79V-Q217H, S18K-V203I, T158A-D259P, N61K-N252K, K43N-Q217R,        Q206R-S260P, V198L-D259G, S161E-S260T, G160A-D259G, A1Y-Q275R,        A200T-H226L, Q217R-T244A, S260P-Q275R, T158I-D259N, L75I-N76D,        S161E-Q185H, Y21H-S37E, S249R-Q275R, N76T-N212D, S260P-Q275L,        G131S-K265N, V4A-S249N, N25D-Q185R, K43R-N76S, S183D-Q206R,        Q10R-Q275K, K43N-S163T, N25D-V26A, S260P-Q275L, K141I-S248N,        L16Q-Q217H, K27R-N269T, P210S-N212D, N118G-V121A, G215D-D259V,        N62Y-G97D, V4E-S260P, K27E-Y91F, Y6D-T55A, N77D-N252T,        N25K-H238R, V44A-Q206H, S37P-S260F, V44A-Q206R,        I31L-S33T-S63G-N109G-A128S-G169A-N218S-N243V,        A1G-I31L-S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-G169A-S224A-N243V-S249Q,        S33T-N61G-S63G-N109G-A128S-G131H-G169A-N218S-N243V,        S33T-S63G-N109G-A128S-G169A-N218S-N243V,        S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V-S249Q,        S33T-N76D-A128S-N218S, N76D-N109G-A128S-S224A, D099N,        K141Y-E156Q, N062L-S063L-Q217L, N062L-S063N, N062L-S063N-Q217E,        N062L-S063R, N062L-S063R-Q217L, N062S-Q217E, N062S-S063G-Q217E,        N062S-S063G-Q217R, N062S-S063N-Q217R, S063G-S125A, D060G-Q217L,        D120N-K141Y-K213Q, K043T-D099N-D120N-K141Y,        K043T-D099N-K141Y-K256Q, K043T-K237A, N062L-S063G-Q217R,        N062L-S063G-S125A, N062L-S063L-Q217E, N062L-S063N-S125A-Q217L,        N062S-Q217R, N062S-S063L-Q217E, N062S-S063R-Q217L, S063G-M222S,        S063G-Q217R, K141Y-D197N, N062L-Q217R, N062L-S063G-Q217L-M222S,        N062L-S063L-Q217R, N062L-S063N-Q217R, N062S-Q217G,        N062S-S063G-Q217G, N062S-S063G-Q217L-M222L,        N062S-S063G-S125A-Q217L, N062S-S063N-Q217E, Q217G,        S033G-N062S-S063G, S063G-Q217G, S063G-Q217L-M222L,        S063G-S125A-Q217R, S063L-Q217R, S063N-M222S, S063N-Q217R,        S063N-S125A-Q217L, S063R-Q217R, S063R-S125A-Q217L,        D099N-E156Q-K256Q, E156Q, K012T-D099N-K213Q, K012T-K256Q,        K043T-D099N-K141Y-K213Q, K043T-E156Q, K141Y-K213Q, N062L-Q217G,        N062L-Q217L-M222L, N062L-Q217L-M222S, N062L-S063G-M222S,        N062L-S063G-Q217L-M222L, N062L-S063G-Q217R-M222S,        N062L-S063N-Q217L-M222S, N062L-S063N-S125A, N062L-S063R-S125A,        N062L-S125A, N062S-S063G-M222S, N062S-S063G-Q217G-M222S,        N062S-S063G-S125A, N062S-S063N-Q217L-M222L,        N062S-S063N-S125A-Q217L, N062S-S063R-Q217G,        N062S-S063R-Q217L-M222S, Q217G-M222S, Q217L-M222S, Q217R,        S033G-S063G-Q217R, S063G-Q217E-M222S, S063G-S125A-Q217G,        S063L-Q217E, S063N-Q217G, S063N-Q217G-M222S, S063N-Q217L-M222S,        S063R-Q217L-M222S and S063R-S125A.-   f) a variant of a parent protease, the variant comprising an amino    acid sequence comprising from three to six amino acid substitutions    selected from the group consisting of X024G/R, X053G, X078N, X101N,    X128A/S, and X217L/Q, wherein the variant has proteolytic activity;-   g) a variant of a protease, the variant having proteolytic activity    and comprising an amino acid sequence which comprises from two to    seven alterations at the amino acid positions corresponding to amino    acid positions of SEQ ID NO:2 selected from the group consisting of    positions 24, 53, 78, 97, 101, 128, and 217, wherein each alteration    is independently    -   (i) an insertion of one or more amino acid residues upstream or        downstream of the amino acid residue which occupies the        position,    -   (ii) a deletion of the amino acid residue which occupies the        position, or    -   (iii) a substitution of the amino acid residue which occupies        the position with a different amino acid residue,-   h) a variant of a parent protease, wherein: the variant comprises an    amino acid sequence having no more than 15 alterations relative to    the parent protease, wherein the alterations are independently    selected from an insertion, a deletion, or a substitution, and the    alterations include a substitution of glycine at positions 24 and    53, a substitution of asparagine at positions 78 and 101, a    substitution of alanine or serine at position 128, and a    substitution of glutamine at position 217; the parent protease has    at least 90% sequence identity to SEQ ID NO:2; and the variant has    increased proteolytic activity relative to the parent protease-   i) a variant of a parent protease, said parent protease at least    90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% sequence identity to SEQ    ID NO:2, said variant having an amino acid sequence having at least    90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% identity to the sequence    of SEQ IDNO:2; said variant comprising an a substitution of glycine    at positions 24 and 53, a substitution of asparagine at positions 78    and 101, a substitution of alanine or serine at position 128, and a    substitution of glutamine at position 217, and said variant having    increased proteolytic activity relative to the parent protease;-   j) mixtures thereof.    and one or more adjunct ingredients, said composition being a fabric    and home care product is disclosed.

In one aspect of said composition, said cold water protease has either aperformance index greater than 1, at least 1.1, at least 1.2, at least1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least1.8, at least 1.9, at least 2 from at least 1 to about 10, from at least1 to about 8 or even from at least 1 to about 5 on BMI at pH 8 and 60°F. when compared to an enzyme having SEQ ID NO:4, as defined in TestMethod 2 or Test Method 3 or said cold water protease has a performanceindex at least 1, at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2 from 1.0 to about 10, from 1.0 to about 8 or even from1.0 to about 5 on BMI at pH 8 and 60° F. when compared to an enzymehaving SEQ ID NO:6, as defined in Test Method 2 or Test Method Sand saidcold water protease is selected from the group consisting of:

-   a) a variant of a parent protease having SEQ ID NO:4, said variant    comprising a group of mutations selected from the following groups    of mutations: P210S, P210S-N218A, S063T-S078N-S101A-S183T-T244N,    N061A-S078N-S224A, S053G-S078N-P129T-Q185T, S063T-S078N-S101A,    S078N-P129T, S063T-S078N-S101A-S183T and S063T-S078N-S101A-T2441;-   b) a variant of a parent protease having SEQ ID NO:2, said variant    comprising a group of mutations selected from the following groups    of mutations: G097A-I111V-M124V-Y217Q, G097A-I111V-Y167A-Y217Q,    S024G-N025G-N061P-G097A-S101N-G128S-Y217Q,    S024G-N025G-S053G-N061P-G097A-S101N-G128A-V203Y-Y217Q,    S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128S-V203Y-Y217Q,    V068A-A092G-Y217Q, N061P-G097A-S101N-G128A-P201S-Y217Q,    S024G-N025G-S053G-N061P-G097A-S101N-G128A-P210S-Y217Q,    S024G-N025G-S053G-N061P-G097A-S101N-G128S-Y217Q,    S024G-N025G-S053G-N061P-S078N-G097A-S101N-I111V-G128S-Y217Q,    S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,    N025G-G097A-S101N-G128A-Y217Q,    N025G-S038G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,    N025G-S053G-N061P-S078N-G128A-Y217Q,    N025G-S053G-N061P-S078N-S101N-G128A-Y217Q,    N025G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,    N025G-S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,    N025G-S078N-G097A-S101N-G128A-Y217Q,    N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,    N025G-T055P-N061P-S078N-S101N-G128A-Y217Q, N061P-S101N-G128A-Y217Q,    S024G-N025G-N061P-G097A-G128A-Y217Q,    S024G-N025G-N061P-G097A-S101N-G128A-Y217Q,    S024G-N025G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,    S024G-N025G-S053G-N061P-S078N-S101N-G128A-Y217Q,    S024G-N025G-S053G-T055P-G097A-S101N-G128A-Y217Q,    S024G-N025G-S053G-T055P-N061P-G128A-Y217Q,    S024G-N025G-T055P-G097A-G128A-Y217Q,    S024G-N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,    S024G-N061P-S078N-G097A-S101N-G128A-Y217Q,    S024G-S053G-N061P-G097A-G128A-Y217Q,    S024G-S053G-N061P-S078N-G097A-G128A-Y217Q,    S024G-S053G-T055P-G097A-S101N-G128A-Y217Q,    S024G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,    S024G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,    S024G-S053G-T055P-N061P-S101N-G128A-Y217Q,    S024G-T055P-N061P-G097A-G128A-Y217Q, S053G-G097A-S101N-G128A-Y217Q,    S053G-N061P-G097A-S101N-G128A-Y217Q-S249N,    S053G-N061P-S078N-G097A-G128A-Y217Q,    S053G-S078N-G097A-S101N-G128A-Y217Q,    S053G-T055P-G097A-S101N-G128A-Y217Q,    S053G-T055P-N061P-S101N-G128A-Y217Q,    S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,    T055P-G097A-S101N-G128A-Y217Q,    T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,    S024G-N025G-N061P-S078N-G097A-S101N-G128A-Y217Q,    S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,    S024G-T055P-N061P-S078N-S101N-G128A-Y217Q,    T055P-N061P-S078N-G128A-Y217Q and S24G-S53G-S78N-S101N-G128A-Y217Q;-   c) a variant of a parent protease having SEQ ID NO:6, said variant    comprising a group of mutations selected from the following groups    of mutations: A116V, G160S, I111L, I115V, N109S, N117M, P005G,    Q059V, T164S, Y262M, A015Q, A015S, A098E, A098N, A098S, A098T,    A098V, A098Y, A114S, A114T, A116G, A116L, A116S, A116T, A116W,    A133G, A133H, A133T, A133V, A137G, A137I, A137L, A137S, A137T,    A138S, A216E, A216F, A216V, D099S, D181E, F261A, F261Q, G024F,    G024I, G024Q, G024Y, G097S, G160T, G211L, G211V, H017F, H017W,    H039V, H226A, I031V, I111V, I268V, K170R, K265R, L016Q, L016T,    L135M, L209T, L209V, L233M, L257T, L257V, L267A, L267V, N025A,    N025I, N025 Q, N025 R, N025 T, N025 V, N101I, N101Q, N101S, N109A,    N109G, N109H, N109L, N109M, N109Q, N109T, N117Q, N184A, N184L,    N184T, N184W, N212G, N212L, N212V, N243P, N252G, N252M, P005T,    P014S, P040G, P040L, P040Q, P129A, P129S, P172G, P172S, P194Q,    P210A, P210S, Q185F, Q185G, Q185I, Q185M, Q185N, Q185S, Q275H,    R186K, S009A, S009G, S009H, S009M, S018T, S130T, S132N, S145K,    S159T, S161I, S161K, S161N, S161T, S162I, S162M, S162Y, S163G,    S182F, S182G, S182V, S182W, S183F, S183L, S183M, S183T, S183V,    S183W, S224A, S236T, S249V, T022A, T022G, T022Q, T022V, T208V,    T242S, T253N, T253S, T254A, T254S, T255L, T255S, T255V, V004A,    V004P, V004W, V084C, V139C, V165M, V203F, Y021K, Y021N, Y021T,    Y021V, Y167F, Y171F, Y214F, Y262F, Y262T, A088T-L257G, A116T-A128S,    N061S-N109G-A128S-N243V-S260P, S009T-N109G-A128S-K141R-N243V,    S009T-S018T-Y021N-N109G-A128S-K141R, S162G-K256R, A088T-N243V,    G024E-A116T, K043Y, N076D-A116T, N218S-S248N, S033T-N243V,    S033T-S063G, S248N-L257G, A001E-S249A, A088T-A116T, A088T-A128S,    A088T-G131H, A088T-N109G, A088T-S248N, A088T-S249A, A116T-N243V,    A116T-T158S, A128S, A128S-K256R, A128S-L257G, A128S-N243V,    A128S-S248N, A128S-T158S, G024E-A088T, G024E-A128S, G024E-G131H,    G024E-K256R, G024E-L257G, G024E-N218S, G024E-N243V, G024E-S162G,    G024E-S249A, G024E-T158S, G131H, G131H-K256R, G131H-S249A,    K043Y-A088T, K043Y-A116T, K256R, N076D-K256R, N109G, N109G-A116T,    N109G-A128S, N109G-A128S-N243V-K256R, N109G-A128S-N243V-S248A,    N109G-G131H, N109G-K256R, N109G-L257G, N109G-N218S, N109G-N243V,    N109G-S248N, N218S-L257G, N243V, N243V-K256R, N243V-L257G,    N243V-S248N, N243V-S249A, Q103H-A128S, Q103H-G131H, Q103H-K256R,    Q103H-L257G, Q103H-N243V, Q103H-S248N, Q103H-S249A, Q103H-T158S,    Q206D-N243V, S033T-A128S, S033T-K256R, S033T-N076D, S033T-N218S,    S033T-S248N, S033T-T158S, S063G-A128S, S063G-K256R, S063G-N243V,    S063G-S162G, S063G-T158S, S248N-K256R, S249A, T158S-N243V,    T158S-S249A, A088T-A116T-N243V-K256R-L257G, A088T-A116T-N243V-L257G,    A088T-T158S-N218S-K256R, A088T-T158S-N218S-N243V-L257G,    A088T-A116T-T158S-N218S-N243V-K256R-L257G,    A088T-N109G-A116T-G131H-A153S-N218S-S248N-L257G,    A088T-N109G-A116T-T158S-S248N-K256R-L257G, A088T-N109G-T158S-L257G,    A114S-A116T-N218S-N243V-S248N-K256R-L257G, A116T-T158S-K256R,    A088T-A116T-G131H-T158S-S248N-L257G, A088T-A116T-T158S,    A088T-N109G-A116T-G131H-L257G,    A088T-N109G-A116T-T158S-N243V-S248N-L257G, A088T-N109G-N243V-L257G,    A088T-N109G-N243V-S248N, A088T-N109G-T158S-N243V-L257G,    A088T-N109G-T158S-N243V-S248N-L257G, A116T-T158S-S248N-L257G,    Y006H-A116T-G131H-S248N, A088T-A116T-G131H-T158S-N218S-N243V,    A088T-A116T-G131H-T158S-N243V,    A088T-A116T-G131H-T158S-N243V-K256R-L257G,    A088T-A116T-N218S-N243V-K256R-L257G, A088T-A116T-S248N-K256R-L257G,    A088T-A116T-T158S-N218S-N243V, A088T-A116T-T158S-N243V-K256R-L257G,    A088T-A116T-T158S-N243V-S248N-L257G,    A088T-G131H-N243V-S248N-K256R-L257G, A088T-N109G-A116T-T158S-L257G,    A088T-N109G-A116T-T158S-N212D-N243V-K256R-L257G,    A088T-N109G-A116T-T158S-N218S-N243V-S248N-K256R,    A088T-N109G-A116T-T158S-S248N-L257G,    A088T-N109G-G131H-V148A-N218S-N243V-K256R-L257G, A088T-N109G-K256R,    A088T-N109G-N243V-S248N-L257G, A088T-N109G-T158S-K256R    A088T-N109G-T158S-N243V, A088T-T158S-N243V-K256R-L257G,    A116T-N218S-N243V-L257G-N269S, A116T-T158S-K256R-L257G,    N109G-A116T-K256R-L257G, N109G-A116T-N243V,    N109G-A116T-T158S-N243V-K256R-L257G, N109G-G131H-L257G,    N109G-G131H-S248N-K256R-L257G, N109G-G131H-T158S-K256R-L257G,    S003P-A116T-T158S-S248N-K256R, T158S-S248N-K256R,    A088T-A116T-G131H-N243V-K256R, A088T-A116T-G131H-S248N-K256R-L257G,    A088T-A116T-G131H-V147A-T158S-N218S-N243V-S248N-L257G,    A088T-A116T-S248N-L257G, A088T-A116T-T158S-N218S,    A088T-A116T-T158S-N218S-K256R-L257G, A088T-A116T-T158S-N218S-L257G,    A088T-G131H-N243V-L257G, A088T-G131H-T158S-S248N-L257G,    A088T-N109G-A116T, A088T-N109G-A116T-G131H-N218S,    A088T-N109G-A116T-G131H-N218S-S248N-L257G,    A088T-N109G-A116T-G131H-N243V-S248N-K256R-L257G,    A088T-N109G-A116T-G131H-T158S-S248N-K256R-L257G,    A088T-N109G-A116T-N218S-N243V-K256R,    A088T-N109G-A116T-N218S-N243V-L257G,    A088T-N109G-A116T-N243V-S248N-K256R,    A088T-N109G-A116T-N243V-S248N-K256R-L257G,    A088T-N109G-A116T-T158S-N243V-L257G,    A088T-N109G-G131H-T158S-N243V-S248N-K256R,    A088T-N109G-G131H-T158S-W241R-S248N-K256R, A088T-N109G-K256R-L257G,    A088T-N109G-L257G, A088T-N109G-N243V, A088T-N109G-N243V-K256R,    A088T-N109G-N243V-K256R-L257G, A088T-N109G-S248N-K256R,    A088T-N109G-T158S-N218S-K256R-L257G,    A088T-N109G-T158S-N218S-N243V-S248N-K256R,    A088T-N109G-T158S-N243V-K256R, A088T-N109G-T158S-N243V-K256R-L257G,    A088T-N109G-T158S-N243V-S248N-A274D, A088T-N109G-T158S-S248N-L257G,    A088T-T158S-K256R, A088T-T158S-N218S-N243V-K256R-L257G,    A088T-T158S-N243V-L257G, A116T-G131H-N218S-N243V-S248N,    A116T-G131H-S248N-L257G, A116T-S248N-K256R-L257G,    A116T-T158S-N218S-N243V-K256R, A116T-T158S-N218S-S248N-L257G-Q271R,    A116T-T158S-N243V-K256R-L257G, A116T-T158S-N243V-S248N-L257G,    G131H-S248N, G131H-T158S-I234T-N243V-K256R,    G131H-W241L-N243V-S248N-K256R,    N109G-A116T-G131H-A137V-T158S-S248N-K256R-L257G,    N109G-A116T-G131H-A151S-N218S-K256R-L257G,    N109G-A116T-G131H-T158S-N218S-N243V-K256R,    N109G-A116T-G131H-T158S-N218S-S248N,    N109G-A116T-G131H-T158S-N243V-S248N, N109G-A116T-S248N,    N109G-A116T-T158S-L257G, N109G-A116T-T158S-N218S-W241R-N243V,    N109G-A116T-T158S-N243V-S248N-L257G,    N109G-A116T-T158S-S248N-K256R-L257G, N109G-A116T-T158S-S248N-L257G,    N109G-G131H-N218S-L257G, N109G-G131H-N218S-S248N-K256R-L257G,    N109G-G131H-T158S-N218S-S248N-K256R-L257G-A274T, N109G-N243V-L257G,    N109G-T158S-N218S-K256R-L257G, N109G-T158S-N218S-L257G,    N109G-T158S-S248N-K256R,    P014L-A015L-L016C-H017T-S018L-Q019K-G020A-Y021T-T022L-G023E,    S003F-A088T-N109G-A116T-T158S-N243V-K256R-L257G,    V004A-A088T-A116T-T158S-N218S,    V004A-N109G-A116T-G131H-S248N-K256R-L257G,    V004L-A116T-N218S-N243V-S248N-L257G, Y006H-N109G-N218S-N243V-S248N,    A001T-A116T-T158S-N243V-L257G, A088T-A116T-G131H-L257G,    A088T-A116T-G131H-N218S-L257G,    A088T-A116T-G131H-N218S-S248N-K256R-L257G,    A088T-A116T-G131H-N218S-S248N-L257G,    A088T-A116T-G131H-N243V-K256R-L257G, A088T-A116T-G131H-N243V-L257G,    A088T-A116T-G131H-N243V-S248N, A088T-A116T-G131H-T158S-K256R-L257G,    A088T-A116T-G131H-T158S-L257G, A088T-A116T-G131H-T158S-N218S,    A088T-A116T-G131H-T158S-N218S-N243V-K256R-A273T,    A088T-A116T-G131H-T158S-N218S-S248N-K256R,    A088T-A116T-G131H-T158S-N218S-S248N-L257G,    A088T-A116T-G131H-T158S-N243V-S248N-K256R,    A088T-A116T-G131H-T158S-S248N, A088T-A116T-K256R,    A088T-A116T-K256R-L257G, A088T-A116T-N218S-N243V-L257G,    A088T-A116T-N243V-K256R, A088T-A116T-N243V-S248N-K256R-L257G,    A088T-A116T-S248N-K256R, A088T-A116T-T158S-K256R,    A088T-A116T-T158S-N218S-N243V-K256R,    A088T-A116T-T158S-N218S-N243V-K256R-N269S,    A088T-A116T-T158S-N218S-N243V-S248N,    A088T-A116T-T158S-N218S-N243V-S248N-K256R-L257G,    A088T-A116T-T158S-N243V-K256R, A088T-A116T-T158S-N243V-L257G,    A088T-A116T-T158S-N243V-S248N-K256R,    A088T-A116T-T158S-N243V-S248N-K256R-L257G,    A088T-A116T-T158S-S248N-K256R, A088T-A116T-V143A-N218S-S248N-K256R,    A088T-A116T-V147I-T158S-N218S-N243V-L257G, A088T-G131H-K256R-L257G,    A088T-G131H-N218S-N243V-S248N, A088T-G131H-N218S-S248N-L257G,    A088T-G131H-S248N-K256R-L257G, A088T-G131H-T158S-L257G,    A088T-G131H-T158S-N218S-K256R,    A088T-G131H-T158S-N218S-N243V-K256R-L257G,    A088T-G131H-T158S-N218S-N243V-L257G, A088T-G131H-T158S-N218S-S248N,    A088T-G131H-T158S-N243V, A088T-G131H-T158S-N243V-S248N,    A088T-G131H-T158S-N243V-S248N-K256R,    A088T-G131H-T158S-N243V-S248N-L257G,    A088T-I107T-N109G-A116T-G131H-T158S-N218S-N243V-S248N,    A088T-N109G-A116T-G131H-N218S-L257G,    A088T-N109G-A116T-G131H-N218S-N243V,    A088T-N109G-A116T-G131H-N218S-N243V-K256R-L257G,    A088T-N109G-A116T-G131H-N218S-N243V-L257G,    A088T-N109G-A116T-G131H-N218S-S248N-K256R-L257G,    A088T-N109G-A116T-G131H-N243V, A088T-N109G-A116T-G131H-N243V-L257G,    A088T-N109G-A116T-G131H-N243V-S248N-L257G,    A088T-N109G-A116T-G131H-S248N, A088T-N109G-A116T-G131H-S248N-K256R,    A088T-N109G-A116T-G131H-S248N-L257G,    A088T-N109G-A116T-G131H-T158S-L257G,    A088T-N109G-A116T-G131H-T158S-N218S,    A088T-N109G-A116T-G131H-T158S-N218S-S248N-K256R,    A088T-N109G-A116T-G131H-T158S-N218T-N243V,    A088T-N109G-A116T-G131H-T158S-N243V-K256R,    A088T-N109G-A116T-G131H-T158S-N243V-K256R-L257G,    A088T-N109G-A116T-G131H-T158S-N243V-S248N,    A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R,    A088T-N109G-A116T-G131H-T158S-S248N-K256R-L257G,    A088T-N109G-A116T-G131H-T158S-S248N-L257G, A088T-N109G-A116T-N218S,    A088T-N109G-A116T-N218S-L257G, A088T-N109G-A116T-N218S-N243V,    A088T-N109G-A116T-N218S-N243V-S248N-L257G,    A088T-N109G-A116T-N218S-S248N-K256R, A088T-N109G-A116T-N218T-K256R,    A088T-N109G-A116T-N218T-K256R-L257G, A088T-N109G-A116T-N243V,    A088T-N109G-A116T-N243V-K256R-L257G,    A088T-N109G-A116T-N243V-K256R-L257G-N269D,    A088T-N109G-A116T-S248N-K256R, A088T-N109G-A116T-T158S,    A088T-N109G-A116T-T158S-N218S-L257G,    A088T-N109G-A116T-T158S-N218S-N243V,    A088T-N109G-A116T-T158S-N218S-N243V-K256R,    A088T-N109G-A116T-T158S-N218S-N243V-K256R-L257G,    A088T-N109G-A116T-T158S-N218S-N243V-L257G,    A088T-N109G-A116T-T158S-N218S-S248N, A088T-N109G-A116T-T158S-N243V,    A088T-N109G-A116T-T158S-N243V-K256R,    A088T-N109G-A116T-T158S-N243V-K256R-L257G, A088T-N109G-G131H-L257G,    A088T-N109G-G131H-N218S-K256R-L257G,    A088T-N109G-G131H-N218S-N243V-K256R,    A088T-N109G-G131H-N218S-N243V-L257G,    A088T-N109G-G131H-N218S-N243V-S248N-K256R-L257G,    A088T-N109G-G131H-N243V, A088T-N109G-G131H-N243V-L257G,    A088T-N109G-G131H-N243V-S248N-K256R,    A088T-N109G-G131H-N243V-S248N-L257G, A088T-N109G-G131H-S248N-L257G,    A088T-N109G-G131H-T158S-L257G,    A088T-N109G-G131H-T158S-N218S-N243V-S248N-K256R,    A088T-N109G-G131H-T158S-N243V, A088T-N109G-G131H-T158S-N243V-K256R,    A088T-N109G-G131H-T158S-N243V-K256R-L257G,    A088T-N109G-G131H-T158S-N243V-L257G, A088T-N109G-N218S-K256R,    A088T-N109G-N218S-N243V-S248N-L257G,    A088T-N109G-N218S-S248N-K256R-L257G,    A088T-N109G-N243V-S248N-K256R-L257G,    A088T-N109G-N243V-S248N-L257G-I268V, A088T-N109G-S248N-K256R-L257G,    A088T-N109G-T158S-N218S-K256R, A088T-N109G-T158S-N218S-N243V-L257G,    A088T-N109G-T158S-N243V-K256R-I268V,    A088T-N109G-T158S-N243V-S248N-Q275R, A088T-N218S-N243V,    A088T-N218S-N243V-S248N-K256R-L257G, A088T-N218S-S248N,    A088T-N218S-S248N-L257G, A088T-N243V-K256R, A088T-N243V-L257G,    A088T-S145T-T158S-S248N, A088T-T158S-L257G,    A088T-T158S-N218S-S248N-L257G, A088T-T158S-N243V-K256R-L257G-Q271H,    A088T-T158S-S248N, A088T-V143A-T158S-K256R, A116T-G131H-K256R,    A116T-G131H-N218S, A116T-G131H-N243V, A116T-G131H-N243V-K256R,    A116T-G131H-N243V-L257G, A116T-G131H-S248N-K256R,    A116T-G131H-T158S-N218S-I234T-N243V-S248N-K256R,    A116T-G131H-T158S-N243V-L257G, A116T-G131H-T158S-N243V-S248N-K256R,    A116T-G131H-V143F-T158S-N218S, A116T-L257G, A116T-N218S,    A116T-N218S-L257G, A116T-N218S-N243V-L257G, A116T-N243V-K256R,    A116T-N243V-S248N, A116T-N243V-S248N-K256R-L257G, A116T-S248N,    A116T-T158S-N218S-N243V, A116T-T158S-N218S-S248N, A116T-T158S-N243V,    A116T-T158S-N243V-K256R, A116T-T158S-N243V-L257G,    A116T-T158S-N243V-S248N, A116T-T158S-S248N-K256R-L257G,    A116T-V149I-T158S-N243V-S248N-K256R-Q271H, G131H-N218S-N243V-L257G,    G131H-N243V, G131H-N243V-S248N-K256R, G131H-T158S,    G131H-T158S-N218S-N243V-K256R, G131H-T158S-N243V-K256R-L257G,    G131H-T158S-N243V-S248N-L257G, N109G-A116T-G131H-N218S-K256R-L257G,    N109G-A116T-G131H-N218S-L257G,    N109G-A116T-G131H-N218S-N243V-K256R-L257G,    N109G-A116T-G131H-N218S-S248N-K256R, N109G-A116T-G131H-N243V-K256R,    N109G-A116T-G131H-N243V-L257G,    N109G-A116T-G131H-N243V-S248N-K256R-L257G, N109G-A116T-G131H-S248N,    N109G-A116T-G131H-S248N-I268V,    N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R,    N109G-A116T-G131H-T158S-N218S-S248N-L257G,    N109G-A116T-G131H-T158S-S248N, N109G-A116T-G131H-T158S-S248N-K256R,    N109G-A116T-N218S, N109G-A116T-N218S-N243V-K256R,    N109G-A116T-N218S-N243V-K256R-L257G, N109G-A116T-N218S-S248N-L257G,    N109G-A116T-N243V-K256R, N109G-A116T-N243V-S248N-K256R-L257G,    N109G-A116T-S248N-L257G, N109G-A116T-T158S-G211V-N243V-S248N-K256R,    N109G-A116T-T158S-K256R-L257G, N109G-A116T-T158S-N218S,    N109G-A116T-T158S-N218S-N243V-K256R-L257G,    N109G-A116T-T158S-N218S-N243V-L257G,    N109G-A116T-T158S-N218S-N243V-S248N-L257G,    N109G-A116T-T158S-N218S-S248N-K256R-L257G, N109G-A116T-T158S-N243V,    N109G-A116T-T158S-Q275R, N109G-G131H-A137V-T158S-N218S-S248N,    N109G-G131H-N218S-K237N, N109G-G131H-N218S-N243V-K256R-L257G,    N109G-G131H-N218S-S248N-K256R, N109G-G131H-N243V-K256R-L257G,    N109G-G131H-S145F-N218S-N243V-K256R-L257G, N109G-G131H-S248N-K256R,    N109G-G131H-S248N-L257G, N109G-G131H-T158S-K256R,    N109G-G131H-T158S-N218S-N243V-K256R, N109G-G131H-T158S-N243V,    N109G-G131H-T158S-N243V-K256R-L257G, N109G-G131H-T158S-N243V-L257G,    N109G-G131H-T158S-S248N-L257G, N109G-G131H-T158S-S248N-Q271R,    N109G-N218S-L257G, N109G-N218S-N243V, N109G-N243V-K256R-L257G,    N109G-N243V-S248N-K256R-L257G, N109G-T158S-I268V, N109G-T158S-K256R,    N109G-T158S-N218S-N243V-K256R-L257G, N109G-T158S-N218S-S248N-L257G,    N109G-T158S-N243V, N109G-T158S-N243V-K256R-L257G,    N109G-T158S-N243V-S248N, N109S-A116T-S248N, N218S,    N218S-N243V-S248N-K256R-L257G, N218S-S248N-L257G, N243V-K256R,    N243V-S248N-K256R, N243V-S248N-K256R-L257G,    S105P-A116T-T158S-N218S-N243V-S248N-K256R, S248N, T158S-N243V-K256R,    T158S-N243V-L257G, S018F-S162L, S018P-D120N, P014T-S037T,    S009T-K141R, S161P-S162L, N61P-S63G-N109Q-A128S-S224A-N243V,    A88T-N109G-A114S-A116T-A128S-N243V,    A88T-N109G-A114S-A116T-A128S-S183L-S224A-N243V, N109G-A128S-S183V,    N109M-A128S-S224A, A88T-N109S-A116T-A128S-S224A-N243V,    N109Q-A128S-S224A-N243V, A88T-N109M-A116T-A128S-S224A-N243V,    S63G-A128S, N109S-A128S-S224A-N243V, A88T-N109G-A116T-N243V,    N61S-N109G-N243V, N101Q-N109Q-A128S-S224A-N243V,    A88T-N109G-A116T-T158S-N243V-K256R, N109G-A116T, A88T-N109G,    N61G-N109G-N243V, N109G-A128S-P129S-S130T-S224A-N243V,    A88T-N109Q-A116T-A128S-S224A-N243V, N062L-S063N-Q217L,    N062S-S063R-Q217E, N062S-S063L-Q217L, S063G, S063G-Q217L, S063N and    S063N-Q217L.

In one aspect of said composition, said variant of a parent protease,said parent protease having at least 60%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identity to SEQ ID NO:2, comprises the amino acidsubstitutions: S024G+S053G+S078N+S101N+G128A/S+Y217Q/L/D; and optionallyone or more groups of subsitutions selected from the following groups ofmutations: A088T+N109G+A116T+G131H+N243V+L257G, S033T+N076D,S009T+N109G+K141R+N243V, S162G+K256R, N109G+A116T, N109G+L257G,S162G+L257G, N061G+N109G+N243V, N109G+N243V+S248A,S033T+N076D+N109G+N218S+N243V+S248N+K256R, N109G+A116T+N243V+K256R,A088T+N109G+A116T+G131H+N243V, A088T+N109G, N109G+N243V, T158S+L257G,N061S+N109G+N243V, P040A+N109G+N243V+S248N+K256R,S009T+S018T+Y021N+N109G+K141R, A088T+N109G+A116T+T158S+N243V+K256R,A088T+N109G+A116T+T158S+N218S+L257G, N109G+K256R, N109G+N243V+K256R,S063G+K256R, S063G+N109G, S063G, S063G+N076D, S033T+N076D+N218S andN076D+N218S.

In one aspect of said composition, said protease is a variant of aparent protease having SEQ ID NO:6, said variant comprising one or moreof the following groups of amino acid substitutions:A088T+N109G+A116T+G131H+N243V+L257G, S033T+N076D,S009T+N109G+A128S+K141R+N243V, S162G+K256R, N109G+A116T, N109G+L257G,S162G+L257G, N061G+N109G+N243V, N109G+A128S+N243V+S248A,S033T+N076D+N109G+A128S+N218S+N243V+S248N+K256R,N109G+A116T+N243V+K256R, A088T+N109G+A116T+G131H+N243V, A088T+N109G,N109G+N243V, T158S+L257G, N061S+N109G+N243V,P040A+N109G+A128S+N243V+S248N+K256R,S009T+S018T+Y021N+N109G+A128S+K141R,A088T+N109G+A116T+T158S+N243V+K256R,A088T+N109G+A116T+T158S+N218S+L257G, N109G+K256R,N109G+A128S+N243V+K256R, S063G+K256R, S063G+N109G, S063G+A128S,S063G+N076D, S033T+N076D+A128S+N218S and N076D+N218S.

In one aspect of said composition, said parent protease is a subtilisinprotease.

In one aspect of said composition, said parent protease is a subtilisinprotease selected from the group consisting of B. amyloliquefacienssubtilisin protease BPN′ (SEQ ID NO:2), Geobacillus stearothermophilus(formerly classified as B. stearothermophilus), B. subtilis, B.licheniformis, B. lentus, B. brevis, B. alkalophilus, B.amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B.coagulans, B. circulans, B. lautus, and B. thuringiensis.

In one aspect of said composition, said cold water protease is a variantof a parent protease, said cold water protease comprising a total ofthree, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14 or 15mutations selected from groups (a) and (b) below, wherein at least onemutation is selected from group (a):

-   a) 1, 9, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 33, 43, 76, 102,    109, 137, 141, 158, 169, 204, 210, 218, 243, 248, 249, 256, 257,    260, and 269; and-   b) 24, 25, 40, 52. 53, 55, 58, 59, 61, 62, 63, 68, 78, 86, 87, 88,    89, 92, 96, 97, 100, 101, 103, 104, 106, 111, 114, 115, 116, 117,    118, 123, 124, 125, 126, 128, 129, 130, 131, 132, 133, 134, 144,    145, 159, 161, 162, 167, 194, 203, 206, 213, 217, 227, 232, 239,    240, 242, 265, 267, and 275.

In one aspect of said composition, said variant of a parent protease,said parent protease having SEQ ID NO:2, comprises a total of three,four, five, six, seven, eight, nine, 10, 11, 12, 13, 14 or 15 mutationsselected from groups (a) and (b) below, wherein at least one mutation isselected from group (a):

-   (a) A1E/T, S9/T, P14L, A15L, L16C, H17T, S18L/T, Q19K, G20A, Y21N/T,    T22L, G23E, S33T, K43Y, N76D, G102A, N109A/S/G, A137V, K141R, T158S,    G169A, S204E, P210S, N218S, N243P/V, S248N/A, S249A, K256R, L257G,    S260P, and N269D; and-   (b) S24G/R/E, N25G, P40A/E, P52L, S53G, T55P, F58G, Q59S,    N61E/P/G/S, N62Q/R/S, S63G/H, V68A, S78N, P86S, S87D/G, A88T/V,    S89Y, A92G, L96T, G97A, G100N/Q/T, S101N, Q103E/H, Y104N, W106F,    I111V, A114G, I115V, A116N/T, N117S, N118G, N123A/G/Q/V, M124I/V,    S125A, L126A, G128A/S, P129E/Q/S/V, S130G, G131S/H, S132N, A133V,    A134T, A144K, S145D, S159K, S161P, S162G/K, Y167A, P194L, V203Y,    Q206D/E, K213L, Y217Q/L/D, V227T, A232T, P239R/V, N240K, T242R,    K265N, L267V, and Q275E.

In one aspect of said composition, said variant of a parent protease hasa total net charge of −1, 0 or +1 relative to the BPN′ wild-type.

In one aspect of said composition, said composition comprises an adjunctingredient selected from the group consisting of: a surfactant, abuilder, a chelating agent, a dye transfer inhibiting agent, adispersant, one or more additional enzymes, an enzyme stabilizer, acatalytic material, a bleach activator, a hydrogen peroxide, a source ofhydrogen peroxide, a preformed peracid, a polymeric dispersing agent, aclay soil removal/anti-redeposition agent, a brightener, a sudssuppressor, a dye, a perfume, a perfume delivery system, a structureelasticizing agent, a fabric softener, a carrier, a hydrotrope, aprocessing aid, a solvent, a pigment and mixtures thereof.

In one aspect of said composition, said composition comprises a materialselected from the group consisting of an encapsulate comprising aperfume, a hueing agent, an amphiphilic cleaning polymer, a brightener,a chelating agent and mixtures thereof.

In one aspect of said composition, said composition comprises a secondnon-immunoequivalent protease selected from the group comprising:

-   a) subtilisins (EC 3.4.21.62);-   b) trypsin-like or chymotrypsin-like proteases;-   c) metalloproteases; and-   d) mixtures thereof.

In one aspect of said composition, said composition comprises a secondnon-immunoequivalent protease that is a subtilisin (EC 3.4.21.62)protease said subtilisin (EC 3.4.21.62) protease being a cold water GG36variant protease.

In one aspect of said composition, said composition comprises anadditional enzyme selected from the group consisting of hemicellulases,peroxidases, proteases, cellulases, cellobiose dehydrogenases,xyloglucanases, xylanases, lipases, phospholipases, esterases,cutinases, pectinases, mannanases, pectate lyases, keratinases,reductases, oxidases, phenoloxidases, lipoxygenases, ligninases,pullulanases, tannases, pentosanases, lichenases glucanases,arabinosidases, hyaluronidase, chondroitinase, laccase, amylases, ormixtures thereof.

In one aspect of said composition, said composition comprises anadditional enzyme is selected from the group consisting of:

-   a) first-wash lipases;-   b) alpha-amylases that are greater than 90% identical to the    wild-type alkaline alpha-amylases derived from Bacillus sp. NCIB    12289, NCIB 12512, NCIB 12513, DSM 9375 DSM 12368, DSMZ no. 12649,    KSM AP1378, KSM K36 or KSM K38;-   c) microbial-derived endoglucanases; and-   d) mixtures thereof.

In one aspect of said composition, said composition comprises a materialselected from the group consisting of:

-   a) an alcohol ethoxysulphate surfactant;-   b) a chelant selected from the group consisting of DTPA (Diethylene    triamine pentaacetic acid), HEDP (Hydroxyethane diphosphonic acid),    DTPMP (Diethylene triamine penta(methylene phosphonic acid)),    ethylenediaminedisuccinic acid (EDDS),    1,2-Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, and    derivatives of such chelants; and-   c) mixtures thereof.

In one aspect of said composition, said composition comprises a materialselected from the group consisting of:

-   a) an alcohol ethoxysulphate surfactant having an alkyl chain length    of from 10 to 14 and a degree of ethoxylation from 1 to 4;-   b) a chelant selected from the group consisting of DTPA (Diethylene    triamine pentaacetic acid), HEDP (Hydroxyethane diphosphonic acid),    DTPMP (Diethylene triamine penta(methylene phosphonic acid)),    ethylenediaminedisuccinic acid (EDDS),    1,2-Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, and    derivatives of such chelants; and-   c) mixtures thereof.

In one aspect of said composition, said composition comprises, based ontotal composition weight, a chelant level of from about 1% to about 5%.

In one aspect of said composition, said composition comprises a fabrichueing agent selected from the group consisting of

-   a) dyes;-   b) dye-clay conjugates comprising at least one cationic-basic dye    and a smectite clay; and-   c) mixtures thereof.

In one aspect of said composition, said composition comprises a fabrichueing agent selected from the group consisting of

-   a) small molecule dyes; polymeric dyes and mixtures thereof;-   b) dye-clay conjugates comprising at least one cationic-basic dye    and a smectite clay; and-   c) mixtures thereof.

In one aspect of said composition, said composition comprises, based ontotal composition weight:

-   a) from about 0.0005 wt % to about 0.1 wt % of said cold water    protease; and-   b) one or more of the following-   (i) from about 0.00003 wt % to about 0.1 wt % fabric hueing agent;-   (ii) from about 0.001 wt % to about 5 wt %, perfume capsules and/or-   (iii) from about 0.1 wt % to about 5 wt % amphiphilic cleaning    polymer.

In one aspect of said composition, said composition has a single ormulti-compartment unit dose form.

In one aspect of said composition, said composition is in the form of amulti-compartment unit dose, wherein at least one protease is in adifferent compartment to any additional enzymes and/or chelant.

In one aspect, a composition comprising a cold water protease and amaterial selected from the group consisting of: an encapsulatecomprising a perfume, a GG36 variant protease, a hueing agent, anamphiphilic cleaning polymer and mixtures thereof said composition beinga fabric and home care product is disclosed.

In one aspect of said composition comprising a cold water protease and amaterial selected from the group consisting of: an encapsulatecomprising a perfume, a GG36 variant protease, a hueing agent, anamphiphilic cleaning polymer and mixtures thereof, said cold waterprotease is a variant of the protease having SEQ ID NO:2 (subtilisinBPN′) said variant comprising one or more of the following sets ofmutations, insertions or deletions: S182E, N109I, N109D-Y217L-S248R,N109D-S188R-Y217L, S87D-Y217L-S248R, S87R-N109D-Y217L-S248R,S87R-N109D-S188D-Y217L-S248R, G128A-Y217Q, I111V-M124V, M124V-Y217Q,N62Q-G97A, S89Y-M124V, V68A, V68A-A92G, V68A-G97A, V68A-I111V,V68A-S89Y, V68A-V227T, V68A-Y217Q, W106F-Y217Q, G97A-G128A-Y217Q,G97A-L126A-Y217Q, G97A-M124V-L126A-Y217Q, G97A-N123G-Y217Q,L96T-G97A-Y217Q, M124V-L126A-Y217Q, N62Q-G128A-Y217Q, N62Q-G97A-Y217Q,G97N-G128A-Y217M, G97G-G128S-Y217E, G97A-G128A-Y217Q, G97M-G128S-Y217E,G97A-G128S-Y217Q, G97D-G128S-Y217Q, G97M-G128G-Y217M, G97G-G128S-Y217Q,G97S-G128S-Y217Q, G97G-G128A-Y217Q, G97S-G128A-Y217E, G97A-G128S-Y217L,G97A-G128A-Y217N, G97Q-G128S-Y217L, G97A-G128A-Y217M, G97A-G128A-Y217S,G97D-G128A-Y217Q, G97M-G128S-Y217Q, G97Q-G128G-Y217D-S87Y,G97S-G128A-Y217N, G97A-G128S-Y217T, G97D-G128S-Y217E, G97D-G128A-Y217L,G97G-G128S-Y217E-S78P-A272T, G97T-G128S-Y217D, G97D-G128A-Y217I,G97Q-G128S-Y217Q, G97G-G128A-Y217D, G97Q-G128A-Y217N, G97S-G128A-Y217M,G97S-G128S-Y217N, G97S-G128S-Y217M, G97E-G128S-Y217M, G97S-G128P-Y217Q,G97T-G128S-Y217Q, G97D-G128S-Y217Q-A73T, G97E-G128S-Y217N,G97G-G128A-Y217I, G97Q-G128A-Y217D, G97Q-G128S-Y217M,G97R-G128T-Y217Q-S162P, G97S-G128S-Y217D, G97T-G128P-Y217I,G97Q-G128G-Y217E, G97C-G128G-Y217N, G97D-G128S-Y217H, G97M-G128S-Y217L,G97M-G128S-Y217N, G97S-G128S-Y217E, G97M-G128S-Y217I, G97A-G128P-Y217A,G97R-G128S-Y217D, G97A-G128A-Y217Q-S145D, G97A-G128A-Y217Q-P239R,G97A-G128A-Y217Q-N61E-P129E-S162K-K213L-N240K, G97A-G128A-Y217Q-N61E,G97A-G128A-Y217Q-P40E-A144K-K213L, G97A-G128A-Y217Q-P129E,G97A-G128A-Y217Q-N61E-P129E-S159K, G97A-G128A-Y217Q-K213L,G97A-G128A-Y217Q-S87D, G97A-G128A-Y217Q-Q206E,G97A-G128A-Y217Q-S24R-P40E-S145D-S159K-K213L, G97A-G128A-Y217Q-K265N,G97A-G128A-Y217Q-S24R, G97A-G128A-Y217Q-P40E, G97A-G128A-Y217Q-Q275E,G97A-G128A-Y217Q-P129E-S145D-N240K, G97A-G128A-Y217Q-A144K,G97A-G128A-Y217Q-S159K, G97A-G128A-Y217Q-S162K, G97A-G128A-Y217Q-N240K,G97A-G128A-Y217Q-S53G, G97A-G128A-Y217Q-S78N,G97A-G128A-Y217Q-S53G-S78N, G97A-G128A-Y217Q-S53G-I111V,G97A-G128A-Y217Q-S53G-N117S, G97A-G128A-Y217Q-S53G-S132N,G97A-G128A-Y217Q-Y104N-S132N, G97A-G128A-Y217Q-S53G-S78N-I111V,G97A-G128A-Y217Q-S53G-S78N-N117S, G97A-G128A-Y217Q-S53G-S78N-S132N,G97A-G128A-Y217Q-S53G-Y104N-S132N, G97A-G128A-Y217Q-S78N-Y104N-S132N,Y217L-V068C-A069G, Y217L-I079F-A098G, Y217L-P086T-S101D-Q103S-V147I,Y217L-A088T-P129S-G146D, Y217L-V093I-G128D-P129R, Y217L-Z096.01D-A098R,Y217L-Z096.01H-A098G, Y217L-G097S-Z097.01S-A098G-A273T,Y217L-A098S-D099G-G100D, Y217L-Z098.01N, Y217L-D099G-Z099.01N,Y217L-D099G-Z099.01S, Y217L-D099V-S101D, Y217L-Z099.01S, Y217L-G100D,Y217L-S101D-Q103H, Y217L-S101G-A151V, Y217L-S101H-G102S,Y217L-S101H-Q103D, Y217L-G102R-Q103C-Y104C-V192I, Y217L-Q103D,Y217L-V121I-I122S-N123C, Y217L-V121L-N123C, Y217L-I122S-N123S,Y217L-M124I, Y217L-M124V, Y217L-L126F-P129Z-S182N, Y217L-L126Y,Y217L-G127S-P129D, Y217L-Z127.01N-G128S-P129S, Y217L-G128H-P129Y,Y217L-G128S-P129D, Y217L-G128S-P129D-S248R, Y217L-G128S-P129G,Y217L-P129G-G131Z, Y217L-P129G-S130H-S132Z, Y217L-P129H-G131Z,Y217L-P129L, Y217L-P129S-S130H-S132Z, Y217L-P129Z, Y217L-P129Z-S130G,Y217L-P129Z-S130G-G131H-S132H, Y217L-P129Z-S130H,Y217L-S130V-G131D-S132I, S87T-A88L-S89G-G97A-G128A-Y217Q,N61P-S63H-G97A-G128A-Y217Q, S87G-A88V-S89A-G97A-G128A-Y217Q,P86S-S87G-A88V-G97A-G128A-Y217Q, Q59S-N61P-G97A-G128A-Y217Q,S24G-N25G-G97A-G128A-Y217Q, N61P-N62S-G97A-G128A-Y217Q,G97A-G128A-P129Q-S130G-G131S-Y217Q, L75S-N76Y-G97A-G128A-Y217Q,G97A-G128A-V203Y-Y217Q, T55P-G97A-G128A-Y217Q,A88V-L90I-G97A-G128A-Y217Q, G97A-G128A-G211R-N212S-K213V-Y217Q,G23A-S24G-N25G-G97A-G128A-Y217Q, T22N-S24A-G97A-G128A-Y217Q,S24R-G97A-G128A-Y217Q, G97A-A98S-G128A-Y217Q,G97A-G128A-T158G-S159G-Y217Q, Q59E-N61P-G97A-G128A-Y217Q,G97A-A98E-G128A-Y217Q,G97A-G128A-Y217Q-P86S-S87G-A88V-A116N-N117S-N118G,G97A-G128A-Y217Q-S63T-P86S-S87G-A88V,G97A-G128A-Y217Q-P86S-S87G-A88V-P239R,G97A-G128A-Y217Q-S24G-N25G-N61P-N62S-P194L-A232T,G97A-G128A-Y217Q-P129Q-S130G-G131S-A133V-L267V,G97A-G128A-Y217Q-A134T-L267V,G97A-G128A-Y217Q-S24R-P40E-P129E-S159K-K265R,G97A-G128A-Y217Q-A134T-G211T, G97A-G128A-Y217Q-S24R-P129E,G97A-G128A-Y217Q-I111V-S161P, G97A-G128A-Y217Q-T55P-P129Q,G97A-G128A-Y217Q-I115V-L267V,G97A-G128A-Y217Q-P86S-S87G-A88V-A116S-N117G-N118R,G97A-G128A-Y217Q-V203Y-L267V,G97A-G128A-Y217Q-S24G-N25G-S78N-S101N-V203Y,G97A-G128A-Y217Q-P52S-T55P-V203Y,G97A-G128A-Y217Q-Q59S-N61P-A116S-N117G-N118R,G97A-G128A-Y217Q-S24G-N25G-P129Q-S130G-G131S,G97A-G128A-Y217Q-P86S-S87G-A88V-T242R, G97A-G128A-Y217Q-P40E-T55P-N269K,G97A-G128A-Y217Q-G23A-S24G-N25G-A116N-N117S-N118G,G97A-G128A-Y217Q-V8L-N25Y-P129Q-S130G-G131S,G97A-G128A-Y217Q-S24G-N25G-S53G-S78N-S87T-A88L-S89G-S101N,G97A-G128A-Y217Q-G211T-L267V, G97A-G128A-Y217Q-S24R-A116N-N117S-N118G,G97A-G128A-Y217Q-S24R-A128S-P129G,G97A-G128A-Y217Q-P129Q-S130G-G131S-N240K,G97A-G128A-Y217Q-N25Y-P129Q-S130G-G131S,G97A-G128A-Y217Q-S87T-A88L-S89G-A134T,G97A-G128A-Y217Q-P129Q-S130G-G131S-L267V,G97A-G128A-Y217Q-S87G-A88V-S89A-A116N-N117S-N118G,G97A-G128A-Y217Q-N61P-P129Q-S130G-G131S,G97A-G128A-Y217Q-N61P-S78N-S87T-A88L-S89G-S101N,G97A-G128A-Y217Q-T55P-P129V-P194S, G97A-G128A-Y217Q-T55P-P129V,G97A-G128A-Y217Q-S24G-N25G-T55P-S78N-S101N,G97A-G128A-Y217Q-T55P-S78N-I115V, G97A-G128A-Y217Q-N25Y-S87G-A88V-S89A,G97A-G128A-Y217Q-A134T-N240K, G97A-G128A-Y217Q-S24R-Q59S-N61P,G97A-G128A-Y217Q-G23A-S24G-N25G-P239R,G97A-G128A-Y217Q-T55P-A116S-N117G-N118R, G97A-G128A-Y217Q-A134T-S161P,G97A-G128A-Y217Q-S24G-N25G-S53G-N61P-S101N-V203Y,G97A-G128A-Y217Q-N25Y-Q59S-N61P,G97A-G128A-Y217Q-N25Y-P129Q-S130G-G131S-A137T,G97A-G128A-Y217Q-G23A-S24G-N25G-N61P-S63H,G97A-G128A-Y217Q-T55P-N61P-S78N-S101N-V203Y,G97A-G128A-Y217Q-P129Q-N240K, G97A-G128A-Y217Q-T55P-A134T,G97A-G128A-Y217Q-N25Y-N61P-S63H, G97A-G128A-Y217Q-S87T-A88L-S89G-P129S,G97A-G128A-Y217Q-T55P-L75H-N76G,G97A-G128A-Y217Q-S24G-N25G-S53G-S78N-S87T-A88L-S89G-S101N-V203Y,G97A-G128A-Y217Q-T55P-I115V, G97A-G128A-Y217Q-T55P-A116N-N117S-N118G,G97A-G128A-Y217Q-S24G-N25G-A116N-N117S-N118G,G97A-G128A-Y217Q-S24R-P129Q-S130G-G131S,G97A-G128A-Y217Q-G23A-S24G-N25G-G211R-N212S-K213V,G97A-G128A-Y217Q-S24G-N25G-T55P-N61P-S78N-S101N-V203Y,G97A-G128A-Y217Q-T55P-S78N-S87T-A88L-S89G-S101N,G97A-G128A-Y217Q-I115V-A273S, G97A-G128A-Y217Q-N25Y-T55P,G97A-G128A-Y217Q-S24G-N25G-S53G-T55P-N61P-S78N-S87T-A88L-S89G-S101N-V203Y,G97A-G128A-Y217Q-Q59S-N61P-N240K, G97A-G128A-Y217Q-S161P-L267V,G97A-G128A-Y217Q-S24G-N25G-S53G-T55P-N61P-S78N-S87T-A88L-S89G-S101N,G97A-G128A-Y217Q-S87T-A88L-S89G-S101N,G97A-G128A-Y217Q-S24G-N25G-N61P-S101N,G97A-G128A-Y217Q-S24G-N25G-S53G-T55P-S101N-V203Y,G97A-G128A-Y217Q-N240K,G97A-G128A-Y217Q-S24G-N25G-S53G-T55P-S87T-A88L-S89G-S101N-V203Y,G97A-G128A-Y217Q-S24G-N25G-T55P-S101N,G97A-G128A-Y217Q-N61P-S63H-A128S-P129Q,G97A-G128A-Y217Q-S89Y-P129Q-S130G-G131S,G97A-G128A-Y217Q-P129Q-S130G-G131S-V203Y, G97A-G128A-Y217Q-I115V-N240K,G97A-G128A-Y217Q-S53G-N61P-S87T-A88L-S89G-S101N-V203Y,G97A-G128A-Y217Q-S161P-V203Y, G97A-G128A-Y217Q-S87T-A88L-S89G-N240K,G97A-G128A-Y217Q-S87T-A88L-S89G-P239R,G97A-G128A-Y217Q-T55P-N61P-S78N-S87T-A88L-S89G-S101N-V203Y,G97A-G128A-Y217Q-S24G-N25G-I115V-A134T,G97A-G128A-Y217Q-Y6Q-P129Q-S130G-G131S, G97A-G128A-Y217Q-T55P-S78N-S89Y,G97A-G128A-Y217Q-S24G-N25G-T55P-S78N-A88V-S101N,G97A-G128A-Y217Q-N61P-S63H-S78N-I111V-A134T,G97A-G128A-Y217Q-S24G-N25G-S53G-T55P-S78N-S101N,G97A-G128A-Y217Q-S24G-N25G-S53G-S78N-S101N-V203Y,G97A-G128A-Y217Q-S53G-N61P-S101N-V203Y,G97A-G128A-Y217Q-S53G-T55P-S78N-S101N-V203Y,G97A-G128A-Y217Q-S53G-T55P-N61P-S78N-S87T-A88L-S89G-S101N,G97A-G128A-Y217Q-N240K-A273S,G97A-G128A-Y217Q-S78N-S87T-A88L-S89G-S101N,G97A-G128A-Y217Q-Q59S-N61P-S87T-A88L-S89G,G97A-G128A-Y217Q-N61P-S63H-S78N-S161P,G97A-G128A-Y217Q-N61P-S63H-S78N-I111V,G97A-G128A-Y217Q-T55P-A128S-P129Q,G97A-G128A-Y217Q-N61P-S78N-S101N-V203Y, G97A-G128A-Y217Q-N61E-A144K,G97A-G128A-Y217Q-A134T-P239R,G97A-G128A-Y217Q-S24G-N25G-S53G-S78N-S87T-A88L-S101N-V203Y,G97A-G128A-Y217Q-S24G-N25G-S53G-T55P-N61P-S101N-V203Y,G97A-G128A-Y217Q-N61P-S78N-S87T-A88L-S89G-S101N-V203Y,G97A-G128A-Y217Q-T55P-N240K,G97A-G128A-Y217Q-S24G-N25G-S87T-A88L-S89G-S101N,G97A-G128A-Y217Q-P129Q-S130G-G131S-P239R, G97A-G128S-Y217Q,G97A-G128A-Y217Q-S53G-N61P-S101N, G97A-G128A-Y217Q-I111V-P129Q-G211T,G97A-G128A-Y217Q-S24G-N25G-S53G-S101N-V203Y,G97A-G128A-Y217Q-Q59S-N61P-S87G-A88V-S89A,G97A-G128A-Y217Q-S24G-N25G-S78N-S87T-A88L-S89G-S101N,G97A-G128A-Y217Q-P129Q-S130G-G131S-S162K,G97A-G128A-Y217Q-T55P-P129Q-S130G-G131S, G97A-G128A-Y217Q-T55P-V203Y,G97A-G128A-Y217Q-S87G-A88V-S89A-P129Q-S130G-G131S,G97A-G128A-Y217Q-S24G-N25G-S53G-T55P-N61P-S78N-S87T-A88L-S89G,G97A-G128A-Y217Q-I111V-P129Q-S130G-G131S, G97A-G128A-Y217Q-I111V-A273S,G97A-G128A-Y217Q-N61P-S87T-A88L-S89G,G97A-G128A-Y217Q-T22N-S24A-N61P-S63H, G97A-G128A-Y217Q,G97A-G128A-Y217Q-S53G-S78N-S87T-A88L-S89G-S101N-P129S-V203Y,G97A-G128A-Y217Q-S159K-L267V,G97A-G128A-Y217Q-P40E-S53Y-S78Y-P86S-S87G-A88V,G97A-G128A-Y217Q-S24R-S145D, G97A-G128A-Y217Q-I111V-S159K,G97A-G128A-Y217Q-T55P-P129L, G97A-G128A-Y217Q-Q59S-N61P-V203Y,G97A-G128A-Y217Q-T55P-S78N-S87T-A88L-S89G-S101N-V203Y,G97A-G128A-Y217Q-S24G-N25G-S53G-S78N-S101N,G97A-G128A-Y217Q-S53G-N61P-S78N-S87T-A88L-S89G-S101N-V203Y,G97A-G128A-Y217Q-S89Y, G97A-G128A-Y217Q-S24R-P129V,G97A-G128A-Y217Q-S87G-A88V-S89A-A116N-N117S-N118G-P172H,G97A-G128A-Y217Q-I111V-A134T,G97A-G128A-Y217Q-Q59S-N61P-P129Q-S130G-G131S,G97A-G128A-Y217Q-P5S-S87G-A88V-S89A-A116G-N117R,Y217Q-N61P-A97G-G102A-A128G-P129S,G97A-G128A-Y217Q-S24G-N25G-S53G-N61P-S78N,G97A-G128A-Y217Q-S145D-S159K-N240K-Q275E,G97A-G128A-Y217Q-T55P-A128S-P129D,G97A-G128A-Y217Q-G23A-S24G-N25G-A128S-P129D,G97A-G128A-Y217Q-S24G-N25G-S53G-S78N-S87T-A88L-S89G-V203Y,G97A-G128A-Y217Q-I111V-P239R, G97A-G128A-Y217Q-S87G-A88V-S89A-S162K,G97A-G128A-Y217Q-S87T-A88L-S89G-I115V,G97A-G128A-Y217Q-S24G-N25G-T55P-S78N, G97A-G128A-Y217Q-T55P-A92G,G97A-G128A-Y217Q-S24G-N25G-S53G-S87T-A88L-S89G-V203Y,G97A-G128A-Y217Q-T22N-S24A-T55P,G97A-G128A-Y217Q-S53G-S87T-A88L-S89G-S101N-V203Y,G97A-G128A-Y217Q-S24G-N25G-S53G-T55P-S78N-S87T-A88L-S89G,G97A-G128A-Y217Q-P129Q-S130G-G131S-S159K,G97A-Y217Q-N61P-N62Q-G100N-A128G,G97A-G128A-Y217Q-S24R-S78N-S182P-L267V, G97A-G128A-Y217Q-P239R-A273S,G97A-G128A-Y217Q-S53G-S78N-S87T-A88L-S89G-S101N-V203Y,G97A-G128A-Y217Q-P129Q-S130G-G131S-T242R,G97A-G128A-Y217Q-S3F-S87T-A88L-S89G-G211T,G97A-G128A-Y217Q-S24G-N25G-L75H-N76G,G97A-G128A-Y217Q-S53G-T55P-N61P-S78N-S87T-A88L-S89G,G97A-G128A-Y217Q-S87T-A88L-S89G-A144K,G97A-G128A-Y217Q-S78N-S87T-A88L-S89G-V203Y,G97A-G128A-Y217Q-Q59S-N61P-A116N-N117S-N118G,G97A-G128A-Y217Q-S87T-A88L-S89G-I111V,G97A-G128A-Y217Q-S24R-S145D-P239R-Q275E, G97A-G128A-Y217Q-S145D-A273S,G97A-G128A-Y217Q-S24G-N25G-K141E-T242R,G97A-G128A-Y217Q-S87T-A88L-S89G-S101N-V203Y,G97A-G128A-Y217Q-A116N-N117S-N118G-P129Q-S130G-G131S,G97A-G128A-Y217Q-S89Y-G211T,G97A-G128A-Y217Q-S87G-A88V-S89A-A116N-N117S-N118G-A144T,G97A-G128A-Y217Q-S24G-N25G-S78N-S87T-A88L-S89G-S101N-V203Y,G97A-G128A-Y217Q-S24G-N25G-P129V, G97A-Y217Q-N61P-A128G-P129S-S130P,G97A-G128A-Y217Q-T55P-N61P-S87T-A88L-S89G-G110C-S130P,G97A-Y217Q-N123G-A128G, G97A-G128A-Y217Q-N61P-N62Q-G100N-G102A-M124I,S78N-G97A-G128A-Y217Q, G97A-S101N-G128A-Y217Q, G97A-G128A-A137V-Y217Q,N61P-G97A-G128A-Y217Q, G97A-G128A-S130P-Y217Q, G97A-Q103N-G128A-Y217Q,S63T-G97A-G128A-Y217Q, G97A-G102A-G128A-Y217Q,G97A-N109D-G128A-Y217Q-S248R, S87R-G97A-G128A-Y217Q,G97A-G128A-S188D-Y217Q, S87D-G97A-G128A-Y217Q-S248R,G97A-G128A-S188D-S248R-Y217Q, G97A-G128A-S248D-Y217Q,S78N-G97A-G128A-Y217Q-L267V, S78N-G97A-G128A-Y217Q-S161P,S78N-G97A-G128A-Y217Q-1115V, S78N-G97A-G128A-Y217Q-A273S,S78N-G97A-G128A-Y217Q-G211T, S78N-G97A-G128A-Y217Q,S78N-G97A-G128A-Y217Q-I111V, S78N-G97A-G128A-Y217Q-V147L,S78N-G97A-G128A-Y217Q-I108V, S78N-G97A-G128A-Y217Q-S89Y,S78N-G97A-G128A-Y217Q-A138T, G97A-G128A-Y217Q-A134T-K213L,G97A-G128A-Y217Q-G23A-S24G-N25G-P129V, G97A-G128A-Y217Q-S24R-P239R,G97A-G128A-Y217Q-S24R-S87T-A88L-S89G

In one aspect of said composition comprising a cold water protease and amaterial selected from the group consisting of: an encapsulatecomprising a perfume, a GG36 variant protease, a hueing agent, anamphiphilic cleaning polymer and mixtures thereof, said variant of theprotease having SEQ ID NO:2 comprises three, four, five, six, seven,eight, nine, 10, 11, 12, 13, 14 or 15 mutations within the group ofpositions comprising positions 24, 25, 40, 52. 53, 55, 58, 59, 61, 62,63, 68, 78, 86, 87, 88, 89, 92, 96, 97, 100, 101, 103, 104, 106, 111,114, 115, 116, 117, 118, 123, 124, 125, 126, 128, 129, 130, 131, 132,133, 134, 144, 145, 159, 161, 162, 167, 194, 203, 206, 213, 217, 227,232, 239, 240, 242, 265, 267, and 275.

In one aspect of said composition comprising a cold water protease and amaterial selected from the group consisting of: an encapsulatecomprising a perfume, a GG36 variant protease, a hueing agent, anamphiphilic cleaning polymer and mixtures thereof, said variant of theprotease having SEQ ID NO:2 comprises a total of three, four, five, six,seven, eight, nine, 10, 11, 12, 13, 14 or 15 mutations selected fromgroups (a) and (b) below:

-   (a) charged mutations selected from the group consisting of N61E,    A144K, P129E, P239R, P40E, Q103E, Q206E, Q275E, S145D, S159K, S162K,    S24R, S63H, S87D and T242R; and-   (b) neutral mutations selected from the group consisting of A114G,    A116N, A133V, A134T, A232T, A88V, A92G, F58G, G100T, G128A, G131S,    G97A, I111V, I115V, K213L, K265N, L126A, L267V, L96T, M124V, N117S,    N118G, N123G, N240K, N25G, N61P, N62Q, N62R, N62S, P129Q, P129V,    P194L, P239V, P52L, P86S, Q59S, S101N, S125A, S130G, S132N, S161P,    S24G, S53G, S78N, S87G, S89Y, T55P, V203Y, V227T, V68A, W106F,    Y104N, Y167A,and Y217Q.

In one aspect of said composition comprising a cold water protease and amaterial selected from the group consisting of: an encapsulatecomprising a perfume, a GG36 variant protease, a hueing agent, anamphiphilic cleaning polymer and mixtures thereof, said variant of theprotease having SEQ ID NO:2 comprises one or more mutations, and havinga total net charge of −1, 0 or +1 relative to the BPN′ wild-type.

In one aspect of said composition comprising a cold water protease and amaterial selected from the group consisting of: an encapsulatecomprising a perfume, a GG36 variant protease, a hueing agent, anamphiphilic cleaning polymer and mixtures thereof, said compositioncomprises an adjunct ingredient selected from the group consisting of: asurfactant, a builder, a chelating agent, a dye transfer inhibitingagent, a dispersant, one or more additional enzyme, an enzymestabilizer, a catalytic material, a bleach activators, a hydrogenperoxide, a source of hydrogen peroxide, a preformed peracid, apolymeric dispersing agent, a clay soil removal/anti-redeposition agent,a brightener, a suds suppressor, a dye, a perfume, a perfume deliverysystem, a structure elasticizing agent, a fabric softener, a carrier, ahydrotrope, a processing aid, a solvent, a pigment and mixtures thereof.

In one aspect of said composition comprising a cold water protease and amaterial selected from the group consisting of: an encapsulatecomprising a perfume, a GG36 variant protease, a hueing agent, anamphiphilic cleaning polymer and mixtures thereof, said compositioncomprises a third non-immunoequivalent protease that is a subtilisin (EC3.4.21.62) protease said subtilisin (EC 3.4.21.62) protease being a coldwater GG36 variant protease.

In one aspect of said composition comprising a cold water protease and amaterial selected from the group consisting of: an encapsulatecomprising a perfume, a GG36 variant protease, a hueing agent, anamphiphilic cleaning polymer and mixtures thereof, said compositioncomprises an additional enzyme selected from the group consisting ofhemicellulases, peroxidases, proteases, cellulases, cellobiosedehydrogenases, xyloglucanases, xylanases, lipases, phospholipases,esterases, cutinases, pectinases, mannanases, pectate lyases,keratinases, reductases, oxidases, phenoloxidases, lipoxygenases,ligninases, pullulanases, tannases, pentosanases, lichenases glucanases,arabinosidases, hyaluronidase, chondroitinase, laccase, amylases, ormixtures thereof.

In one aspect of said composition comprising a cold water protease and amaterial selected from the group consisting of: an encapsulatecomprising a perfume, a GG36 variant protease, a hueing agent, anamphiphilic cleaning polymer and mixtures thereof, said compositioncomprises an additional enzyme is selected from the group consisting of:

-   a) first-wash lipases;-   b) alpha-amylases that are greater than 90% identical to the    wild-type alkaline alpha-amylases derived from Bacillus sp. NCIB    12289, NCIB 12512, NCIB 12513, DSM 9375 DSM 12368, DSMZ no. 12649,    KSM AP1378, KSM K36 or KSM K38;-   c) microbial-derived endoglucanases; and-   d) mixtures thereof.

In one aspect of said composition comprising a cold water protease and amaterial selected from the group consisting of: an encapsulatecomprising a perfume, a GG36 variant protease, a hueing agent, anamphiphilic cleaning polymer and mixtures thereof, said compositioncomprises a material selected from the group consisting of:

-   a) an alcohol ethoxysulphate surfactant;-   b) a chelant selected from the group consisting of DTPA (Diethylene    triamine pentaacetic acid), HEDP (Hydroxyethane diphosphonic acid),    DTPMP (Diethylene triamine penta(methylene phosphonic acid)),    ethylenediaminedisuccinic acid (EDDS),    1,2-Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, and    derivatives of such chelants; and-   c) mixtures thereof.

In one aspect of said composition comprising a cold water protease and amaterial selected from the group consisting of: an encapsulatecomprising a perfume, a GG36 variant protease, a hueing agent, anamphiphilic cleaning polymer and mixtures thereof, said compositioncomprises a material selected from the group consisting of:

-   a) an alcohol ethoxysulphate surfactant having an alkyl chain length    of from 10 to 14 and a degree of ethoxylation from 1 to 4;-   b) a chelant selected from the group consisting of DTPA (Diethylene    triamine pentaacetic acid), HEDP (Hydroxyethane diphosphonic acid),    DTPMP (Diethylene triamine penta(methylene phosphonic acid)),    ethylenediaminedisuccinic acid (EDDS),    1,2-Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, and    derivatives of such chelants; and-   c) mixtures thereof.

In one aspect of said composition comprising a cold water protease and amaterial selected from the group consisting of: an encapsulatecomprising a perfume, a GG36 variant protease, a hueing agent, anamphiphilic cleaning polymer and mixtures thereof, said compositioncomprises, based on total composition weight, a chelant level of fromabout 1% to about 5%.

In one aspect of said composition comprising a cold water protease and amaterial selected from the group consisting of: an encapsulatecomprising a perfume, a GG36 variant protease, a hueing agent, anamphiphilic cleaning polymer and mixtures thereof, said compositioncomprises a fabric hueing agent selected from the group consisting of

-   a) dyes;-   b) dye-clay conjugates comprising at least one cationic-basic dye    and a smectite clay; and-   c) mixtures thereof.

In one aspect of said composition comprising a cold water protease and amaterial selected from the group consisting of: an encapsulatecomprising a perfume, a GG36 variant protease, a hueing agent, anamphiphilic cleaning polymer and mixtures thereof, said compositioncomprises a fabric hueing agent selected from the group consisting of

-   a) small molecule dyes; polymeric dyes and mixtures thereof;-   b) dye-clay conjugates comprising at least one cationic-basic dye    and a smectite clay; and-   c) mixtures thereof.

In one aspect of said composition comprising a cold water protease and amaterial selected from the group consisting of: an encapsulatecomprising a perfume, a GG36 variant protease, a hueing agent, anamphiphilic cleaning polymer and mixtures thereof, said compositioncomprises based on total composition weight:

-   a) from about 0.0005 wt % to about 0.1 wt % of said cold water    protease; and-   b) one or more of the following:    -   (i) from about 0.00003 wt % to about 0.1 wt % of a fabric hueing        agent;    -   (ii) from about 0.001 wt % to about 5 wt %, of a perfume        capsules;    -   (iii) from about 0.1 wt % to about 5 wt % of a amphiphilic        cleaning polymer;    -   (iv) from about 0.00003 wt % to about 0.1 wt % of a GG36 variant        protease.

In one aspect of said composition comprising a cold water protease and amaterial selected from the group consisting of: an encapsulatecomprising a perfume, a GG36 variant protease, a hueing agent, anamphiphilic cleaning polymer and mixtures thereof, said composition hasa single or multi-compartment unit dose form.

In one aspect of said composition comprising a cold water protease and amaterial selected from the group consisting of: an encapsulatecomprising a perfume, a GG36 variant protease, a hueing agent, anamphiphilic cleaning polymer and mixtures thereof, said composition isin the form of a multi-compartment unit dose, wherein at least oneprotease is in a different compartment to any additional enzymes and/orchelant.

A fabric and home product that may comprise one or more cold waterproteases and a material selected from the group consisting of: anencapsulate comprising a perfume, a hueing agent, an amphiphiliccleaning polymer and mixtures thereof, with the balance of any aspectsof the aforementioned composition is made up of one or more adjunctmaterials, is disclosed.

In one aspect of the aforementioned fabric and home care product, saidfabric and home care product may comprise, based on total fabric andhome care product weight, from about 0.0005 wt % to about 0.1 wt %, fromabout 0.001 wt % to about 0.05 wt %, or even from about 0.002 wt % toabout 0.03 wt % of said cold water protease.

In one aspect of the aforementioned fabric and home care product, saidfabric and home care product may comprise, based on total fabric andhome care product weight, about 0.00003 wt % to about 0.1 wt %, fromabout 0.00008 wt % to about 0.05 wt %, or even from about 0.0001 wt % toabout 0.04 wt %, fabric hueing agent;

In one aspect of the aforementioned fabric and home care product, saidfabric and home care product may comprise, based on total fabric andhome care product weight, from about 0.001 wt % to about 5 wt %, fromabout 0.01 wt % to about 2 wt %, or even from about 0.03 wt % to about0.5 wt %, perfume capsules.

In one aspect of the aforementioned fabric and home care product, saidfabric and home care product may comprise, based on total fabric andhome care product weight, from about 0.1 wt % to about 5 wt %, fromabout 0.25 wt % to about 2.5 wt %, or even from about 0.3 wt % to about1.5 wt % amphiphilic cleaning polymer.

Cold Water Proteases

In addition to the cold water proteases disclosed above in the Fabricand Home Care Products, suitable cold water protease variants areenzymes that exhibit one or more of the following four criteria:

-   -   (a) a performance index of at least 1.1, at least 1.2, at least        1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at        least 1.8, at least 1.9; at least 2 from 1.1 to about 10, from        1.1 to about 8 or even from 1.1 to about 5 on BMI at pH 8 and        60° F. when compared to Purafect Prime (SEQ ID NO:2 with the        mutation Y217L), as defined in Test Method 2 and/or Test Method        3;    -   (b) a performance index of at least 1.3, at least 1.4, at least        1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at        least 2, from 1.3 to about 10, from 1.3 to about 8 or even from        1.3 to about 5 on BMI at pH 8 and 60° F. when compared to BPN′        (SEQ ID NO:2), as defined in Test Method 2 and/or Test Method 3;    -   (c) a performance index of at least 0.9, at least 1.0, at least        1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at        least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2        from 0.9 to about 10, from 0.9 to about 8 or even from 0.9 to        about 5 on BMI at pH 8 and 60° F. when compared to SEQ ID NO:4,        as defined in Test Method 2 and/or Test Method 3;    -   (d) a performance index of at least 0.9, at least 1.0, at least        1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at        least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2        from 0.9 to about 10, from 0.9 to about 8 or even from 0.9 to        about 5 on BMI at pH 8 and 60° F. when compared to SEQ ID NO:6,        as defined in Test Method 2 and/or Test Method 3.

Examples of such proteases can be found in Examples 2-18 and 31 of thepresent specification. Cold water proteases as defined above may be usedin Fabric and Home Care Products. Thus, in one aspect, Fabric and HomeCare Products comprising such cold water proteases are disclosed.

GG36 Proteases of this Invention

Suitable GG36 proteases can be selected from the variants described inExamples 19 to 30. In one aspect, GG36 cold water proteases may beSeries 1 GG36 cold water proteases.

Suitable Series 1 GG36 cold water protease variants include enzymesderived from a parent protease, said parent protease's sequence being atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5% or 100% identical to the amino acid sequenceof SEQ ID NO:755, said variant having one or more of the followingcharacteristics:

-   -   a) a Test Method 4 performance index of at least 1.1, at least        1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at        least 1.7, at least 1.8, at least 1.9; at least 2; from 1.1 to        about 10, from 1.1 to about 8 or even from 1.1 to about 5;    -   b) a Test Method 5 performance index of at least 1.1, at least        1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at        least 1.7, at least 1.8, at least 1.9, at least 2; from 1.1 to        about 10, from 1.1 to about 8 or even from 1.1 to about 5;    -   c) a Test Method 6 performance index of at least 1.0, at least        1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at        least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2,        from 1.0 to about 10, from 1.0 to about 8 or even from 1.0 to        about 5.    -   Suitable Series 1 GG36 cold water proteases can be derived from        subtilisins, particularly those derived from subtilisin Bacillus        Lentus of SEQ ID NO:755 and in one aspect may comprise one or        more of the following mutations:    -   A1R, Q2S, V4R, V4S, S9A, R10S, P14K, A16S, H17R, N18R, G20R,        T22A, T22R, S24R, S24W, G25R, G25V, V26F, L42I, N43R, N43A,        G46R, P52F, P52E, P52N, T57R, Q59A, N62E, N62Q, V68A, V68C,        T71G, I72C, A74C. L75A, L75F, L75R, N76D, S78R, L82R, P86W,        E89P, E89T, E89G, E89H, E89I, E89V, E89W, Y91N, K94N, G100S,        S101A, S101N, S101G, S101D, S103G, S103N, V104L, V104I, S106V,        S106G, A108I, L111V, E112V, G115K, G115R, N117F, G118I, V121F,        S128D, S128F, S128L, S128N, P129E, S144R, L148I, A158E. G159E,        S160D, S166D, N185E, N185I, R186H, S188E, S188D, D197F, V203E,        Y209S, Y209N, Y209F, Y209T, Y209E, Y209H, Y209G, P210R, S212I,        S212F, Y214F, A215N, A215D, A215E, L217E, L217N, T224A, A230E,        A231I, Q236F, N238R, N238K, P239K, P239G, P239R, P239S, W241R,        S242R, S242L, N243R, V244R, N248I, N248V, H249R, L250I, N252R,        T253R, L262D, Y263F, S265F, L267V, L267N. N269I, N269R, E271F,        E271I, E271H, E271P, E271T, E271V, E271L and/or A272F    -   In one aspect, suitable Series 1 GG36 cold water proteases        include subtilisins, particularly Bacillus Lentus of SEQ ID        NO:755, that may comprise one or more of the following sets of        mutations, insertions or deletions:    -   T022R-S024R, S009A-E271L, N018R-W241R, N018R-G115R, N043R-H249R,        G020R-H249R, V004R-H249R, G020R-S024R, N018R-H249R, S009A-G020R,        G020R-W241R, S009A-S078R, G020R-G115R, N018R-S024R, S024R-S242R,        T022R-G115R, N018R-N043R, G020R-N043R, N018R-S242R, S242R-N269R,        N018R-V244R, S024R-N269R, G020R-E271L, S024R-E271L, V004R-S009A,        G020R-N269R, A001R-S024R, V244R-E271L, S009A-N018R, W241R-E271L,        V004R-S024R, S009A-H249R, S009A-T022R, N062E-P129E, N062E-G159E,        A016S-L148I, A158E-H249R, A016S-N062E, L111V-S188D, T022A-N062E,        N062E-L148I, T022A-P129E, N062E-E271F, N062E-A158E, A016S-G159E,        N062E-R186H, S128N-G159E, N062E-S188D, N062E-S128N, L148I-G159E,        S103G-A158E, L111V-G159E, A158E-E271F, A016S-S188D, T022A-L111V,        S128N-A158E, A016S-A158E, V104L-A158E, S128N-R186H, G159E-Y209E,        N062E-S101A, L111V-Y209E, L148I-S188D, S101A-Y209E, T022A-S188D,        A016S-T022A, S128N-P129E, A016S-Y209E, A016S-S128N, T022A-E089P,        S128N-Y209E, E089P-A158E, N062E-S103G, R186H-E271F, A016S-P129E,        E089P-G159E, L111V-H249R, S101A-P129E, L148I-Y209E, T022A-G159E,        P129E-H249R, P129E-Y209E, V104L-P129E, S128N-S188D, L111V-A158E,        T022A-A158E, N062E-Y209E, N062E-H249R, S101A-R186H, E089P-P129E,        P129E-E27I, T22A-L111V-G159E, S101A-S103G-V104L-Y209E,        S101A-S103G-V104L-G159E, S101A-S103G-V104L-S188D,        S101G-S103A-V104I-G159D, T22A-S103G-G159E,        T22A-S128N-E271F-Y209E, T22A-Y209E-E271F, T22A-S101A-Y209E,        S101A-Y209E-E271F, T22A-L111V-S128N, T22A-S101A-G159E,        S101A-S103G-V104L, T22A-S101A-S103G-V104L, S101A-S103G-V104L,        S101G-S103A-V104I, S101A-S103G-V104L-S128N,        S103A-V104I-G159D-A232V-Q236H-Q245R-N248D-N252K,        S101G-V104I-G159D-A232V-Q236H-Q245R-N248D-N252K,        S101G-S103A-G159D-A232V-Q236H-Q245R-N248D-N252K,        S101G-S103A-V104L-A232V-Q236H-Q245R-N248D-N252K,        S101G-S103A-V104L-G159D-Q236H-Q245R-N248D-N252K,        S101G-S103A-V104L-G159D-A232V-Q245R-N248D-N252K,        S101G-S103A-V104L-G159D-A232V-Q236H-N248D-N252K,        S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N252K,        S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N248D,        N62E-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F,        N62E-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-H249R,        T22A-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-H249R,        S101G-S103A-V104I-G159D-A232V-Q245R-N248D-S24R,        S101G-S103A-V104I-G159D-A232V-Q245R-N248D-T253R,        S101G-S103A-V104I-A158E-A232V-Q245R-N248D-H249R,        T22A-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F,        S101G-S103A-V104I-G159E-A232V-Q245R-N248D-H249R,        S101G-S103A-V104I-G159D-A232V-Q245R-N248D-N238R,        S101G-S103A-V104I-A158E-A232V-Q245R-N248D-E271F,        S101G-S103A-V104I-G159D-A232V-Q245R-N248D,        S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F,        S101G-S103A-V104I-G159D-A232V-Q245R-N248D-N76D, and/or        S101G-S103A-V104I-G159E-A232V-Q245R-N248D-E271F.    -   In one aspect, suitable Series 1 GG36 cold water proteases        include variants of subtilisins, particularly Bacillus Lentus of        SEQ ID NO:755, said variants comprising three, four, five, six,        seven, eight, nine, 10, 11, 12, 13, 14 or 15 mutations within        the group of positions comprising positions 1, 2, 4, 9, 10, 14,        16, 17, 18, 20, 22, 24, 25, 26, 42, 43, 46, 52, 57, 59, 62, 68,        71, 72, 74, 75, 76, 78, 82, 86, 89, 91, 94, 100, 101, 103, 104,        106, 108, 111, 112, 115, 117, 118, 121, 128, 129, 144, 148, 158,        159, 160, 166, 185, 186, 188, 197, 203, 209, 210, 212, 214, 215,        217, 224, 230, 231, 236, 238, 239, 241, 242, 243, 244, 248, 249,        250, 252, 253, 262, 263, 265, 267, 269, 271 and 272.    -   In one aspect, suitable Series 1 GG36 cold water proteases        include variants of subtilisins, particularly Bacillus Lentus of        SEQ ID NO:755, said variants comprising a total of three, four,        five, six, seven, eight, nine, 10, 11, 12, 13, 14 or 15        mutations selected from the group comprising: A1R, Q2S, V4R,        V4S, S9A, R10S, P14K, A16S, H17R, N18R, G20R, T22A, T22R, S24R,        S24W, G25R, G25V, V26F, L42I, N43R, N43A, G46R, P52F, P52E,        P52N, T57R, Q59A, N62E, N62Q, V68A, V68C, T71G, I72C, A74C.        L75A, L75F, L75R, N76D, S78R, L82R, P86W, E89P, E89T, E89G,        E89H, E89I, E89V, E89W, Y91N, K94N, G100S, S101A, S101N, S101G,        S101D, S103G, S103N, V104L, V104I, S106V, S106G, A108I, L111V,        E112V, G115K, G115R, N117F, G118I, V121F, S128D, S128F, S128L,        S128N, P129E, S144R, L148I, A158E. G159E, S160D, S166D, N185E,        N185I, R186H, S188E, S188D, D197F, V203E, Y209S, Y209N, Y209F,        Y209T, Y209E, Y209H, Y209G, P210R, S212I, S212F, Y214F, A215N,        A215D, A215E, L217E, L217N, T224A, A230E, A231I, Q236F, N238R,        N238K, P239K, P239G, P239R, P239S, W241R, S242R, S242L, N243R,        V244R, N248I, N248V, H249R, L250I, N252R, T253R, L262D, Y263F,        S265F, L267V, L267N, N269I, N269R, E271F, E271I, E271H, E271P,        E271T, E271V, E271L and A272F; and optionally one or more of the        following mutations: S103A, G159D, Q236H, Q245R, N248D and        N252K.

In one aspect, said Series 1 GG36 cold water protease is variant ofsubtilisin GG36 having SEQ ID NO:755, said variant comprising one ormore mutations, and having a total net charge of −1, −4, −3, −2, −1 or 0relative to subtilisin GG36 wild-type, is disclosed.

In one aspect, said Series 1 GG36 cold water proteases are low ionicstrength cold water proteases. Such low ionic strength proteases arevariants of subtilisin GG36 having SEQ ID NO:755, said variantscomprising one or more mutations, and having a total net charge of −5,−4, −3, −2, −1 or 0 relative to subtilisin GG36 wild-type, is disclosed.These mutations are selected from:

-   -   (a) two or more of the following mutations: A1R, Q2S, V4R, V4S,        S9A, R10S, P14K, A16S, T22A, T22R, S24R, G25V, V26F, L42I, P52F,        P52E, P52N, N62E, N62Q, V68A, V68C, T71G, I72C, A74C, L75A,        L75F, S78R, E89P, E89T, E89G, E89H, E89W, Y91N, K94N, G100S,        S101A, S101N, S101G, S101D, S103G, S103N, V104L, V104I, A108I,        L111V, E112V, G115K, N117F, V121F, S128D, S128F, S128L, S128N,        P129E, L148I, A158E. G159E, S160D, S166D, N185E, R186H, S188E,        S188D, V203E, Y209S, Y209N, Y209F, Y209T, Y209E, Y209H, Y209G,        P210R, S212I, S212F, Y214F, A215N, A215D, A215E, L217E, L217N,        T224A, A230E, A231I, Q236F, N238R, N238K, P239K, P239G, P239R,        N248V, H249R, L250I, L262D, Y263F, S265F, L267V, L267N. N269I,        N269R, E271F, E271I, E271H and A272F; and/or    -   (b) one or more of the following sets of mutations: N062E-P129E,        N062E-G159E, A016S-L148I, A158E-H249R, A016S-N062E, L111V-S188D,        T022A-N062E, N062E-L148I, T022A-P129E, N062E-E271F, N062E-A158E,        A016S-G159E, N062E-R186H, S128N-G159E, N062E-S188D, N062E-S128N,        L148I-G159E, S103G-A158E, L111V-G159E, A158E-E271F, A016S-S188D,        T022A-L111V, S128N-A158E, A016S-A158E, V104L-A158E, S128N-R186H,        G159E-Y209E, N062E-S101A, L111V-Y209E, L148I-S188D, S101A-Y209E,        T022A-S188D, A016S-T022A, S128N-P129E, A016S-Y209E, A016S-S128N,        T022A-E089P, S128N-Y209E, E089P-A158E, N062E-S103G, R186H-E271F,        A016S-P129E, E089P-G159E, L111V-H249R, S101A-P129E, L148I-Y209E,        T022A-G159E, P129E-H249R, P129E-Y209E, V104L-P129E, S128N-S188D,        L111V-A158E, T022A-A158E, N062E-Y209E, N062E-H249R, S101A-R186H,        E089P-P129E, P129E-E271F, T22A-L111V-G159E,        S101A-S103G-V104L-Y209E, S101A-S103G-V104L-G159E,        S101A-S103G-V104L-S188D, S101G-S103A-V104I-G159D,        T22A-S103G-G159E, T22A-S128N-E271F-Y209E, T22A-Y209E-E271F,        T22A-S101A-Y209E, S101A-Y209E-E271F, T22A-L111V-S128N,        T22A-S101A-G159E, S101A-S103G-V104L, T22A-S101A-S103G-V104L,        S101A-S103G-V104L, S101G-S103A-V104I, S101A-S103G-V104L-S128N,        S103A-V104I-G159D-A232V-Q236H-Q245R-N248D-N252K,        S101G-V104I-G159D-A232V-Q236H-Q245R-N248D-N252K,        S101G-S103A-G159D-A232V-Q236H-Q245R-N248D-N252K,        S101G-S103A-V104L-A232V-Q236H-Q245R-N248D-N252K,        S101G-S103A-V104L-G159D-Q236H-Q245R-N248D-N252K,        S101G-S103A-V104L-G159D-A232V-Q245R-N248D-N252K,        S101G-S103A-V104L-G159D-A232V-Q236H-N248D-N252K,        S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N252K,        S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N248D,        N62E-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F,        N62E-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-H249R,        T22A-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-H249R,        S101G-S103A-V104I-G159D-A232V-Q245R-N248D-S24R,        S101G-S103A-V104I-G159D-A232V-Q245R-N248D-T253R,        S101G-S103A-V104I-A158E-A232V-Q245R-N248D-H249R,        T22A-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F,        S101G-S103A-V104I-G159E-A232V-Q245R-N248D-H249R,        S101G-S103A-V104I-G159D-A232V-Q245R-N248D-N238R,        S101G-S103A-V104I-A158E-A232V-Q245R-N248D-E271F,        S101G-S103A-V104I-G159D-A232V-Q245R-N248D,        S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F,        S101G-S103A-V104I-G159D-A232V-Q245R-N248D-N76D and        S101G-S103A-V104I-G159E-A232V-Q245R-N248D-E271F;

In one aspect the above low ionic strength Series 1 GG36 cold waterproteases form part of a detergent composition that is diluted in water,typically within a washing machine, to form a wash liquor, whoseconductivity is from about 0.1 mS/cm to about 3 mS/cm, from about 0.3mS/cm to about 2.5 mS/cm, or even from about 0.5 mS/cm to about 2 mS/cm

In one aspect, said Series 1 GG36 cold water proteases are high ionicstrength cold water proteases. Such high ionic strength proteases arevariants of subtilisin GG36 having SEQ ID NO:755, said variantscomprising two or more mutations, and having a total net charge of +5,+4, +3, +2, +1 or 0 relative to subtilisin GG36 wild-type. Thesemutations are selected from:

-   -   a) two or more of the following mutations V4R, H17R, N18R, G20R,        T22R, S24R, S24W, G25R, N43R, N43A, G46R, P52F, P52N, T57R,        Q59A, N62Q, T71G, L75R, N76D, S78R, L82R, P86W, E89P, E89W,        E89T, E89I, E89H, E89V, V104L, S106V, S106G, G115R, G118I,        V121F, S144R, N185I, D197F, Y209N, Y209S, L217E, A231I, P239R,        P239S, W241R, S242R, S242L, N243R, V244R, N248I, H249R, N252R,        T253R, E271T, E271V, E271L, E271H, E271F, E271P, A1R, S9A, S212F        and N269R; and/or    -   b) one or more of the following sets of mutations T022R-S024R,        S009A-E271L, N018R-W241R, N018R-G115R, N043R-H249R, G020R-H249R,        V004R-H249R, G020R-S024R, N018R-H249R, S009A-G020R, G020R-W241R,        S009A-S078R, G020R-G115R, N018R-S024R, S024R-S242R, T022R-G115R,        N018R-N043R, G020R-N043R, N018R-S242R, S242R-N269R, N018R-V244R,        S024R-N269R, G020R-E271L, S024R-E271L, V004R-S009A, G020R-N269R,        A001R-S024R, V244R-E271L, S009A-N018R, W241R-E271L, V004R-S024R,        S009A-H249R, S009A-T022R,        S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F,        S101G-S103A-V104I-A158E-A232V-Q245R-N248D-E271F,        S101G-S103A-V104I-A158E-A232V-Q245R-N248D-H249R,        S101G-S103A-V104I-G159D-A232V-Q245R-N248D-S24R,        S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N252K,        S101G-S103A-V104L-A232V-Q236H-Q245R-N248D-N252K

In one aspect, the above high ionic strength Series 1 GG36 cold waterproteases form part of a detergent composition that is diluted in water,typically within a washing machine, to form a wash liquor, whoseconductivity is from about 3 mS/cm to about 30 mS/cm, from about 3.5mS/cm to about 20 mS/cm, or even from about 4 mS/cm to about 10 mS/cm.

The charge of the Series 1 GG36 cold water protease variants isexpressed relative to subtilisin GG36 protease wild-type having theamino acid sequence of SEQ ID NO:755. The amino acids that impart asingle negative charge are D and E and those that impart a singlepositive charge are R, H and K. Any amino acid change versus SEQ IDNO:755 that changes a charge is used to calculate the charge of theSeries 1 GG36 cold water protease variant. For example, introducing anegative charge mutation from a wild-type neutral position will add anet charge of −1 to the Series 1 GG36 cold water protease variant,whereas introducing a negative charge mutation (D or E) from a wild-typepositive amino acid residue (R, H or K) will add a net charge of −2.Summing the charge changes from all the amino acid residues that aredifferent for the Series 1 GG36 cold water protease variant versussubtilisin GG36 protease wild-type having the amino acid sequence of SEQID NO:755 gives the charge change of the Series 1 GG36 cold waterprotease variant. Without wishing to be bound by theory, it is believedthat:

(a) the preferred charge range for Series 1 GG36 cold water proteases tobe used in low conductivity laundry detergent solutions is −5, −4, −3,−2, −1, 0, particularly −2, −1;

(b) the preferred charge range for Series 1 GG36 cold water proteases tobe used in high conductivity laundry detergent solutions is +5, +4, +3,+2, +1, 0, particularly +2, +1. By correctly selecting the chargeunexpectedly improved levels of cold water cleaning performance can beobtained.

Low conductivity solutions are defined as having a conductivity of fromabout 0.1 mS/cm to about 3 mS/cm, from about 0.3 mS/cm to about 2.5mS/cm, or even from about 0.5 mS/cm to about 2 mS/cm.

High conductivity having conductivity solutions are defined as having aconductivity of from about 3 mS/cm to about 30 mS/cm, from about 3.5mS/cm to about 20 mS/cm, or even from about 4 mS/cm to about 10 mS/cm

The above examples should be viewed as non-limiting. Once mutations arecombined to optimize cold water performance, the enzyme charge can alsobe balanced by mutations in further positions.

Protease Amino Acid Numbering, Enzyme Nomenclature and AdditionalDefinitions

The numbering of amino acid positions used in this patent is theBacillus amyloliquefaciens subtilisin BPN′ numbering system. Each aminoacid position of each protease variant, including each Series 1 GG36cold water protease variant, is numbered according to the numbering ofcorresponding amino acid position in the amino acid sequence of Bacillusamyloliquefaciens subtilisin BPN′ as determined by alignment of thevariant protease amino acid sequence with the Bacillus amyloliquefacienssubtilisin BPN′ amino acid sequence.

An alternative numbering scheme is numbering the specific amino acidsequence of the B. lentus subtilisin GG36 protease, having the aminoacid sequence of SEQ ID NO:755. None of the amino acid positions of theprotease variants, including the Series 1 GG36 cold water proteasevariants, described herein are numbered using this alternative numberingscheme.

In describing Series 1 GG36 cold water protease variants herein, thefollowing nomenclature is used for ease of reference: Original aminoacid(s):position(s):substituted amino acid(s).

Mutations are named by the one letter code for the parent amino acid,followed by a three digit position number and then the one letter codefor the variant amino acid. For example, mutating glycine (G) atposition 87 to serine (S) is represented as “G087S” or “G875”. Multiplemutations are indicated by inserting a “-” between the mutations.Mutations at positions 87 and 90 are represented as either “G087S-A090Y”or “G87S-A90Y” or “G87S+A90Y” or “G087S+A090Y”. For deletions, the oneletter code “Z” is used. For an insertion relative to the parentsequence, the one letter code “Z” is on the left side of the positionnumber. For a deletion, the one letter code “Z” is on the right side ofthe position number. For insertions, the position number is the positionnumber before the inserted amino acid(s), plus 0.01 for each amino acid.For example, an insertion of three amino acids alanine (A), serine (S)and tyrosine (Y) between position 87 and 88 is shown as“Z087.01A-Z087.02S-Z087.03Y.” Thus, combining all the mutations aboveplus a deletion at position 100 is:“G087S-Z087.01A-Z087.02S-Z087.03Y-A090Y-A100Z.”

In all cases, the accepted IUPAC single letter or triple letter aminoacid abbreviation is employed. The single letter X refers to any of thetwenty amino acids.

The term “wild-type” in reference to an amino acid sequence or nucleicacid sequence indicates that the amino acid sequence or nucleic acidsequence is native or naturally occurring sequence. As used herein, theterm “naturally-occurring” refers to anything (e.g., proteins, aminoacids, or nucleic acid sequences) that are found in nature (i.e., havenot been manipulated by means of recombinant methods). As used herein,the term, “non-naturally occurring” refers to anything that is not foundin nature (e.g., recombinant nucleic acids produced in the laboratory).

As used herein with regard to amino acid residue positions,“corresponding to” or “corresponds to” or “corresponds” refers to anamino acid residue at the enumerated position in a protein or peptide,or an amino acid residue that is analogous, homologous, or equivalent toan enumerated residue in a protein or peptide. As used herein,“corresponding region” generally refers to an analogous position alongrelated proteins or a reference protein.

The terms “derived from” and “obtained from” refer to not only aprotease produced or producible by a strain of the organism in question,but also a protease encoded by a DNA sequence isolated from such strainand produced in a host organism containing such DNA sequence.Additionally, the term refers to a protease which is encoded by a DNAsequence of synthetic and/or cDNA origin and which has the identifyingcharacteristics of the protease in question. To exemplify, “proteasesderived from Bacillus” refers to those enzymes having proteolyticactivity which are naturally-produced by Bacillus, as well as to serineproteases like those produced by Bacillus sources but which through theuse of genetic engineering techniques are produced by non-Bacillusorganisms transformed with a nucleic acid encoding the serine proteases.

The term “identical” in the context of two nucleic acids or polypeptidesequences refers to the residues in the two sequences that are the samewhen aligned for maximum correspondence, as measured using one of thefollowing sequence comparison or analysis algorithms.

As used herein, “homologous genes” refers to a pair of genes fromdifferent, but usually related species, which correspond to each otherand which are identical or very similar to each other. The termencompasses genes that are separated by speciation (i.e., thedevelopment of new species) (e.g., orthologous genes), as well as genesthat have been separated by genetic duplication (e.g., paralogousgenes).

The term “mature” form of a protein, polypeptide, or peptide refers tothe functional form of the protein, polypeptide, or peptide without thesignal peptide sequence and propeptide sequence.

As used herein, “homology” refers to sequence similarity or identity,with identity being preferred. Homology may be determined using standardtechniques known in the art (See e.g., Smith and Waterman, Adv. Appl.Math. 2:482 [1981]; Needleman and Wunsch, J. Mol. Biol. 48:443 [1970\;Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]; softwareprograms such as GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package (Genetics Computer Group, Madison, Wis.); andDevereux et al., Nucl. Acid Res. 12:387-395 [1984]). One example of auseful algorithm is PILEUP. PILEUP creates a multiple sequence alignmentfrom a group of related sequences using progressive, pair-wisealignments. It can also plot a tree showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng and Doolittle (See, Feng andDoolittle, J. Mol. Evol. 35:351-360 [1987]). The method is similar tothat described by Higgins and Sharp (See, Higgins and Sharp, CABIOS5:151-153 [1989]). Useful PILEUP parameters including a default gapweight of 3.00, a default gap length weight of 0.10, and weighted endgaps. Another example of a useful algorithm is the BLAST algorithm,described by Altschul et al., (See, Altschul et al., J. Mol. Biol.215:403-410 [1990]; and Karlin and Altschul, Proc. Natl. Acad. Sci. USA90:5873-5787 [1993]). A particularly useful BLAST program is theWU-BLAST-2 program (See, Altschul et al., Meth. Enzymol. 266:460-480[1996]). WU-BLAST-2 uses several search parameters, most of which areset to the default values. The adjustable parameters are set with thefollowing values: overlap span=1, overlap fraction=0.125, word threshold(T)=11. The HSP S and HSP S2 parameters are dynamic values and areestablished by the program itself depending upon the composition of theparticular sequence and composition of the particular database againstwhich the sequence of interest is being searched. However, the valuesmay be adjusted to increase sensitivity.

The percent sequence identity between a reference sequence and a testsequence of interest may be readily determined by one skilled in theart. The percent identity shared by polynucleotide or polypeptidesequences is determined by direct comparison of the sequence informationbetween the molecules by aligning the sequences and determining theidentity by methods known in the art. An example of an algorithm that issuitable for determining sequence similarity is the BLAST algorithm,(See, Altschul, et al., J. Mol. Biol., 215:403-410 [1990]). Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence that either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. These initial neighborhood word hitsact as starting points to find longer HSPs containing them. The wordhits are expanded in both directions along each of the two sequencesbeing compared for as far as the cumulative alignment score can beincreased. Extension of the word hits is stopped when: the cumulativealignment score falls off by the quantity X from a maximum achievedvalue; the cumulative score goes to zero or below; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLAST programuses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix(See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 [1992])alignments (B) of 50, expectation (E) of 10, M′5, N′−4, and a comparisonof both strands.

The BLAST algorithm then performs a statistical analysis of thesimilarity between two sequences (See e.g., Karlin and Altschul, supra).One measure of similarity provided by the BLAST algorithm is thesmallest sum probability (P(N)), which provides an indication of theprobability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a nucleic acid isconsidered similar to a serine protease nucleic acid of this inventionif the smallest sum probability in a comparison of the test nucleic acidto a serine protease nucleic acid is less than about 0.1, morepreferably less than about 0.01, and most preferably less than about0.001. Where the test nucleic acid encodes a serine proteasepolypeptide, it is considered similar to a specified serine proteasenucleic acid if the comparison results in a smallest sum probability ofless than about 0.5, and more preferably less than about 0.2.

Percent “identical” or “identity” in the context of two or more nucleicacid or polypeptide sequences refers to two or more sequences that arethe same or have a specified percentage of nucleic acid residues oramino acid residues, respectively, that are the same, when compared andaligned for maximum similarity, as determined using a sequencecomparison algorithm or by visual inspection. “Percent sequenceidentity” or “% identity” or “% sequence identity or “% amino acidsequence identity” of a subject amino acid sequence to a reference(i.e., query) amino acid sequence means that the subject amino acidsequence is identical (i.e., on an amino acid-by-amino acid basis) by aspecified percentage to the query amino acid sequence over a comparisonlength when the sequences are optimally aligned. Thus, 80% amino acidsequence identity or 80% identity with respect to two amino acidsequences means that 80% of the amino acid residues in two optimallyaligned amino acid sequences are identical.

“Percent sequence identity” or “% identity” or “% sequence identity or“% nucleotide sequence identity” of a subject nucleic acid sequence to areference (i.e. query) nucleic acid sequence means that the subjectnucleic acid sequence is identical (i.e., on a nucleotide-by-nucleotidebasis for a polynucleotide sequence) by a specified percentage to thequery sequence over a comparison length when the sequences are optimallyaligned. Thus, 80% nucleotide sequence identity or 80% identity withrespect to two nucleic acid sequences means that 80% of the nucleotideresidues in two optimally aligned nucleic acid sequences are identical.

In some embodiments, the percent sequence identity or % sequenceidentity”or “% identity” of a subject sequence to a query sequence canbe calculated by optimally aligning the two sequences and comparing thetwo optimally aligned sequences over the comparison length. The numberof positions in the optimal alignment at which identical residues occurin both sequences are determined, thereby providing the number ofmatched positions, and the number of matched positions is then dividedby the total number of positions of the comparison length (which, unlessotherwise specified, is the length of the query sequence). The resultingnumber is multiplied by 100 to yield the percent sequence identity ofthe subject sequence to the query sequence.

“Optimal alignment” or “optimally aligned” refers to the alignment oftwo (or more) sequences giving the highest percent identity score. Forexample, optimal alignment of two protein sequences can be achieved bymanually aligning the sequences such that the maximum number ofidentical amino acid residues in each sequence are aligned together orby using software programs or procedures described herein or known inthe art. Optimal alignment of two nucleic acid sequences can be achievedby manually aligning the sequences such that the maximum number ofidentical nucleotide residues in each sequence are aligned together orby using software programs or procedures described herein or known inthe art.

In some embodiments, two polypeptide sequences are deemed “optimallyaligned” when they are aligned using defined parameters, such as adefined amino acid substitution matrix, gap existence penalty (alsotermed gap open penalty), and gap extension penalty, so as to achievethe highest similarity score possible for that pair of sequences. TheBLOSUM62 scoring matrix (See, Henikoff and Henikoff, supra) is oftenused as a default scoring substitution matrix in polypeptide sequencealignment algorithms (e.g., BLASTP). The gap existence penalty isimposed for the introduction of a single amino acid gap in one of thealigned sequences, and the gap extension penalty is imposed for eachresidue position in the gap. Exemplary alignment parameters employedare: BLOSUM62 scoring matrix, gap existence penalty=11, and gapextension penalty=1. The alignment score is defined by the amino acidpositions of each sequence at which the alignment begins and ends (e.g.,the alignment window), and optionally by the insertion of a gap ormultiple gaps into one or both sequences, so as to achieve the highestpossible similarity score.

Optimal alignment between two or more sequences can be determinedmanually by visual inspection or by using a computer, such as, but notlimited to for example, the BLASTP program for amino acid sequences andthe BLASTN program for nucleic acid sequences (See e.g., Altschul etal., Nucleic Acids Res. 25(17):3389-3402 (1997); See also, the NationalCenter for Biotechnology Information (NCBI) website).

Suitable Fabric Hueing Agents

Fluorescent optical brighteners emit at least some visible light. Incontrast, fabric hueing agents can alter the tint of a surface as theyabsorb at least a portion of the visible light spectrum. Suitable fabrichueing agents include dyes, dye-clay conjugates, and pigments thatsatisfy the requirements of Test Method 1 in the Test Method Section ofthe present specification. Suitable dyes include small molecule dyes andpolymeric dyes. Suitable small molecule dyes include small molecule dyesselected from the group consisting of dyes falling into the Colour Index(C.I.) classifications of Direct Blue, Direct Red, Direct Violet, AcidBlue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, ormixtures thereof, for example:

(1) Tris-Azo Direct Blue Dyes of the Formula

where at least two of the A, B and C napthyl rings are substituted by asulfonate group, the C ring may be substituted at the 5 position by anNH₂ or NHPh group, X is a benzyl or naphthyl ring substituted with up to2 sulfonate groups and may be substituted at the 2 position with an OHgroup and may also be substituted with an NH₂ or NHPh group.(2) Bis-Azo Direct Violet Dyes of the Formula:

where Z is H or phenyl, the A ring is preferably substituted by a methyland methoxy group at the positions indicated by arrows, the A ring mayalso be a naphthyl ring, the Y group is a benzyl or naphthyl ring, whichis substituted by sulfate group and may be mono or disubstituted bymethyl groups.(3) Blue or Red Acid Dyes of the Formula

where at least one of X and Y must be an aromatic group. In one aspect,both the aromatic groups may be a substituted benzyl or naphthyl group,which may be substituted with non water-solubilising groups such asalkyl or alkyloxy or aryloxy groups, X and Y may not be substituted withwater solubilising groups such as sulfonates or carboxylates. In anotheraspect, X is a nitro substituted benzyl group and Y is a benzyl group(4) Red Acid Dyes of the Structure

where B is a naphthyl or benzyl group that may be substituted with nonwater solubilising groups such as alkyl or alkyloxy or aryloxy groups, Bmay not be substituted with water solubilising groups such as sulfonatesor carboxylates.(5) Dis-Azo Dyes of the Structure

wherein X and Y, independently of one another, are each hydrogen, C₁-C₄alkyl or C₁-C₄-alkoxy, Rα is hydrogen or aryl, Z is C₁-C₄ alkyl;C₁-C₄-alkoxy; halogen; hydroxyl or carboxyl, n is 1 or 2 and m is 0, 1or 2, as well as corresponding salts thereof and mixtures thereof(6) Triphenylmethane Dyes of the Following Structures

and mixtures thereof.

In another aspect, suitable small molecule dyes include small moleculedyes selected from the group consisting of Colour Index (Society ofDyers and Colourists, Bradford, UK) numbers 1,4-Naphthalenedione,1-[2-[2-[4-[[4-(acetyloxy)butyl]ethylamino]-2-methylphenyl]diazenyl]-5-nitro-3-thienyl]-Ethanone,1-hydroxy-2-(1-naphthalenylazo)-Naphthalenedisulfonic acid, ion(2-),1-hydroxy-2-[[4-(phenylazo)phenyl]azo]-Naphthalenedisulfonic acid,ion(2-),2-[(1E)-[4-[bis(3-methoxy-3-oxopropyl)amino]-2-methylphenyl]azo]-5-nitro-3-Thiophenecarboxylicacid, ethyl ester,2-[[4-[(2-cyanoethyl)ethylamino]phenyl]azo]-5-(phenylazo)-3-Thiophenecarbonitrile,2-[2-[4-[(2-cyanoethyl)ethylamino]phenyl]diazenyl]-5-[2-(4-nitrophenyl)diazenyl]-3-Thiophenecarbonitrile,2-hydroxy-1-(1-naphthalenylazo)-Naphthalenedisulfonic acid, ion(2-),2-hydroxy-1-[[4-(phenylazo)phenyl]azo]-Naphthalenedisulfonic acid,ion(2-),4,4′-[[4-(dimethylamino)-2,5-cyclohexadien-1-ylidene]methylene]bis[N,N-dimethyl-Benzenamine,6-hydroxy-5-[(4-methoxyphenyl)azo]-2-Naphthalenesulfonic acid,monosodium salt, 6-hydroxy-5-[(4-methylphenyl)azo]-2-Naphthalenesulfonicacid, monosodium salt,7-hydroxy-8-[[4-(phenylazo)phenyl]azo]-1,3-Naphthalenedisulfonic acid,ion(2-),7-hydroxy-8-[2-(1-naphthalenyl)diazenyl]-1,3-Naphthalenedisulfonic acid,ion(2-),8-hydroxy-7-[2-(1-naphthalenyl)diazenyl]-1,3-Naphthalenedisulfonic acid,ion(2-),8-hydroxy-7-[2-[4-(2-phenyldiazenyl)phenyl]diazenyl]-1,3-Naphthalenedisulfonicacid, ion(2-), Acid Black 1, Acid black 24, Acid Blue 113, Acid Blue 15,Acid Blue 17, Acid Blue 25, Acid blue 29, Acid blue 3, Acid blue 40,Acid blue 45, Acid blue 62, Acid blue 7, Acid blue 75, Acid Blue 80,Acid Blue 83, Acid blue 9, Acid Blue 90, Acid green 27, Acid orange 12,Acid orange 7, Acid red 14, Acid red 150,Acid red 151, Acid red 17, Acidred 18, Acid red 266, Acid red 27, Acid red 4, Acid red 51, Acid red 52,Acid red 73, Acid red 87, Acid red 88, Acid red 92, Acid red 94, Acidred 97, Acid Violet 15, Acid Violet 17, Acid Violet 24, Acid violet 43,Acid Violet 49, Basic blue 159, Basic blue 16, Basic blue 22, Basic blue3, Basic blue 47, Basic blue 66, Basic blue 75, Basic blue 9, Basicviolet 1, Basic violet 2, Basic violet 3, Basic violet 4, Basic violet10, Basic violet 35, C.I. Acid black 1, C.I. Acid Blue 10, C.I. AcidBlue 113, C.I. Acid Blue 25, C.I. Acid Blue 29, C.I. Acid Blue 290 C.I.Acid Red 103, C.I. Acid red 150, C.I. Acid red 52, C.I. Acid red 73,C.I. Acid red 88, C.I. Acid red 91, C.I. Acid violet 17, C.I. Acidviolet 43, C.I. Direct Blue 1, C.I. Direct Blue 120, C.I. Direct Blue34, C.I. Direct Blue 70, C.I. Direct Blue 71, C.I. Direct Blue 72, C.I.Direct Blue 82, C.I. Direct violet 51, C.I. Disperse Blue 10, C.I.Disperse Blue 100, C.I. Disperse Blue 101, C.I. Disperse Blue 102, C.I.Disperse Blue 106:1, C.I. Disperse Blue 11, C.I. Disperse Blue 12, C.I.Disperse Blue 121, C.I. Disperse Blue 122, C.I. Disperse Blue 124, C.I.Disperse Blue 125, C.I. Disperse Blue 128, C.I. Disperse Blue 130, C.I.Disperse Blue 133, C.I. Disperse Blue 137, C.I. Disperse Blue 138, C.I.Disperse Blue 139, C.I. Disperse Blue 142, C.I. Disperse Blue 146, C.I.Disperse Blue 148, C.I. Disperse Blue 149, C.I. Disperse Blue 165, C.I.Disperse Blue 165:1, C.I. Disperse Blue 165:2, C.I. Disperse Blue 165:3,C.I. Disperse Blue 171, C.I. Disperse Blue 173, C.I. Disperse Blue 174,C.I. Disperse Blue 175, C.I. Disperse Blue 177, C.I. Disperse Blue 183,C.I. Disperse Blue 187, C.I. Disperse Blue 189, C.I. Disperse Blue 193,C.I. Disperse Blue 194, C.I. Disperse Blue 200, C.I. Disperse Blue 201,C.I. Disperse Blue 202, C.I. Disperse Blue 205, C.I. Disperse Blue 206,C.I. Disperse Blue 207, C.I. Disperse Blue 209, C.I. Disperse Blue 21,C.I. Disperse Blue 210, C.I. Disperse Blue 211, C.I. Disperse Blue 212,C.I. Disperse Blue 219, C.I. Disperse Blue 220, C.I. Disperse Blue 222,C.I. Disperse Blue 224, C.I. Disperse Blue 225, C.I. Disperse Blue 248,C.I. Disperse Blue 252, C.I. Disperse Blue 253, C.I. Disperse Blue 254,C.I. Disperse Blue 255, C.I. Disperse Blue 256, C.I. Disperse Blue 257,C.I. Disperse Blue 258, C.I. Disperse Blue 259, C.I. Disperse Blue 260,C.I. Disperse Blue 264, C.I. Disperse Blue 265, C.I. Disperse Blue 266,C.I. Disperse Blue 267, C.I. Disperse Blue 268, C.I. Disperse Blue 269,C.I. Disperse Blue 270, C.I. Disperse Blue 278, C.I. Disperse Blue 279,C.I. Disperse Blue 281, C.I. Disperse Blue 283, C.I. Disperse Blue 284,C.I. Disperse Blue 285, C.I. Disperse Blue 286, C.I. Disperse Blue 287,C.I. Disperse Blue 290, C.I. Disperse Blue 291, C.I. Disperse Blue 294,C.I. Disperse Blue 295, C.I. Disperse Blue 30, C.I. Disperse Blue 301,C.I. Disperse Blue 303, C.I. Disperse Blue 304, C.I. Disperse Blue 305,C.I. Disperse Blue 313, C.I. Disperse Blue 315, C.I. Disperse Blue 316,C.I. Disperse Blue 317, C.I. Disperse Blue 321, C.I. Disperse Blue 322,C.I. Disperse Blue 324, C.I. Disperse Blue 328, C.I. Disperse Blue 33,C.I. Disperse Blue 330, C.I. Disperse Blue 333, C.I. Disperse Blue 335,C.I. Disperse Blue 336, C.I. Disperse Blue 337, C.I. Disperse Blue 338,C.I. Disperse Blue 339, C.I. Disperse Blue 340, C.I. Disperse Blue 341,C.I. Disperse Blue 342, C.I. Disperse Blue 343, C.I. Disperse Blue 344,C.I. Disperse Blue 345, C.I. Disperse Blue 346, C.I. Disperse Blue 351,C.I. Disperse Blue 352, C.I. Disperse Blue 353, C.I. Disperse Blue 355,C.I. Disperse Blue 356, C.I. Disperse Blue 357C.I. Disperse Blue 358,C.I. Disperse Blue 36, C.I. Disperse Blue 360, C.I. Disperse Blue 366,C.I. Disperse Blue 368, C.I. Disperse Blue 369, C.I. Disperse Blue 371,C.I. Disperse Blue 373, C.I. Disperse Blue 374, C.I. Disperse Blue 375,C.I. Disperse Blue 376, C.I. Disperse Blue 378, C.I. Disperse Blue 38,C.I. Disperse Blue 42, C.I. Disperse Blue 43, C.I. Disperse Blue 44,C.I. Disperse Blue 47, C.I. Disperse Blue 79, C.I. Disperse Blue 79:1,C.I. Disperse Blue 79:2, C.I. Disperse Blue 79:3, C.I. Disperse Blue 82,C.I. Disperse Blue 85, C.I. Disperse Blue 88, C.I. Disperse Blue 90,C.I. Disperse Blue 94, C.I. Disperse Blue 96, C.I. Disperse Violet 10,C.I. Disperse Violet 100, C.I. Disperse Violet 102, C.I. Disperse Violet103, C.I. Disperse Violet 104, C.I. Disperse Violet 106, C.I. DisperseViolet 107, C.I. Disperse Violet 12, C.I. Disperse Violet 13, C.I.Disperse Violet 16, C.I. Disperse Violet 2, C.I. Disperse Violet 24,C.I. Disperse Violet 25, C.I. Disperse Violet 3, C.I. Disperse Violet33, C.I. Disperse Violet 39, C.I. Disperse Violet 42, C.I. DisperseViolet 43, C.I. Disperse Violet 45, C.I. Disperse Violet 48, C.I.Disperse Violet 49, C.I. Disperse Violet 5, C.I. Disperse Violet 50,C.I. Disperse Violet 53, C.I. Disperse Violet 54, C.I. Disperse Violet55, C.I. Disperse Violet 58, C.I. Disperse Violet 6, C.I. DisperseViolet 60, C.I. Disperse Violet 63, C.I. Disperse Violet 66, C.I.Disperse Violet 69, C.I. Disperse Violet 7, C.I. Disperse Violet 75,C.I. Disperse Violet 76, C.I. Disperse Violet 77, C.I. Disperse Violet82, C.I. Disperse Violet 86, C.I. Disperse Violet 88, C.I. DisperseViolet 9, C.I. Disperse Violet 91, C.I. Disperse Violet 92, C.I.Disperse Violet 93, C.I. Disperse Violet 93:1, C.I. Disperse Violet 94,C.I. Disperse Violet 95, C.I. Disperse Violet 96, C.I. Disperse Violet97, C.I. Disperse Violet 98, C.I. Disperse Violet 99, C.I. ReactiveBlack 5, C.I. Reactive Blue 19, C.I. Reactive Blue 4, C.I. Reactive Red2, C.I. Solvent Blue 43, C.I. Solvent Blue 43, C.I. Solvent Red 14, C.I.Acid black 24, C.I. Acid blue 113, C.I. Acid Blue 29, C.I. Direct violet7, C.I. Food Red 14, Dianix Violet CC, Direct blue 1, Direct Blue 71,Direct blue 75, Direct blue 78, Direct blue 80, Direct blue 279, Directviolet 11, Direct violet 31, Direct violet 35, Direct violet 48, Directviolet 5, Direct Violet 51, Direct violet 66, Direct violet 9, DisperseBlue 106, Disperse blue 148, Disperse blue 165, Disperse Blue 3,Disperse Blue 354, Disperse Blue 364, Disperse blue 367, Disperse Blue56, Disperse Blue 77, Disperse Blue 79, Disperse blue 79:1, Disperse Red1, Disperse Red 15, Disperse Violet 26, Disperse Violet 27, DisperseViolet 28, Disperse violet 63, Disperse violet 77, Eosin Y, Ethanol,2,2′-[[4-[(3,5-dinitro-2-thienyl)azo]phenyl]imino]bis-, diacetate(ester), Lumogen F Blue 650, Lumogen F Violet 570,N-[2-[2-(3-acetyl-5-nitro-2-thienyl)diazenyl]-5-(diethylamino)phenyl]-Acetamide,N-[2-[2-(4-chloro-3-cyano-5-formyl-2-thienyl)diazenyl]-5-(diethylamino)phenyl]-Acetamide,N-[5-[bis(2-methoxyethyl)amino]-2-[2-(5-nitro-2,1-benzisothiazol-3-yl)diazenyl]phenyl]-Acetamide,N-[5-[bis[2-(acetyloxy)ethyl]amino]-2-[(2-bromo-4,6-dinitrophenyl)azo]phenyl]-Acetamide,Naphthalimide, derivatives, Oil Black 860, Phloxine B, Pyrazole, RoseBengal, Sodium6-hydroxy-5-(4-isopropylphenylazo)-2-naphthalenesulfonate, Solvent Black3, Solvent Blue 14, Solvent Blue 35, Solvent Blue 58, Solvent Blue 59,Solvent Red 24, Solvent Violet 13, Solvent Violet 8, Sudan Red 380,Triphenylmethane, and Triphenylmethane, derivatives or mixtures thereof.

Suitable polymeric dyes include polymeric dyes selected from the groupconsisting of polymers containing conjugated chromogens (dye-polymerconjugates) and polymers with chromogens co-polymerized into thebackbone of the polymer and mixtures thereof.

In another aspect, suitable polymeric dyes include polymeric dyesselected from the group consisting of fabric-substantive colorants soldunder the name of Liquitint® (Milliken, Spartanburg, S.C., USA),dye-polymer conjugates formed from at least one reactive dye and apolymer selected from the group consisting of polymers comprising amoiety selected from the group consisting of a hydroxyl moiety, aprimary amine moiety, a secondary amine moiety, a thiol moiety andmixtures thereof. In still another aspect, suitable polymeric dyesinclude polymeric dyes selected from the group consisting of Liquitint®(Milliken, Spartanburg, S.C., USA) Violet CT, carboxymethyl cellulose(CMC) conjugated with a reactive blue, reactive violet or reactive reddye such as CMC conjugated with C.I. Reactive Blue 19, sold by Megazyme,Wicklow, Ireland under the product name AZO-CM-CELLULOSE, product codeS-ACMC, alkoxylated triphenyl-methane polymeric colourants, alkoxylatedthiophene polymeric colourants, and mixtures thereof.

Suitable dye clay conjugates include dye clay conjugates selected fromthe group comprising at least one cationic/basic dye and a smectiteclay, and mixtures thereof. In another aspect, suitable dye clayconjugates include dye clay conjugates selected from the groupconsisting of one cationic/basic dye selected from the group consistingof C.I. Basic Yellow 1 through 108, C.I. Basic Orange 1 through 69, C.I.Basic Red 1 through 118, C.I. Basic Violet 1 through 51, C.I. Basic Blue1 through 164, C.I. Basic Green 1 through 14, C.I. Basic Brown 1 through23, CI Basic Black 1 through 11, and a clay selected from the groupconsisting of Montmorillonite clay, Hectorite clay, Saponite clay andmixtures thereof. In still another aspect, suitable dye clay conjugatesinclude dye clay conjugates selected from the group consisting of:Montmorillonite Basic Blue B7 C.I. 42595 conjugate, MontmorilloniteBasic Blue B9 C.I. 52015 conjugate, Montmorillonite Basic Violet V3 C.I.42555 conjugate, Montmorillonite Basic Green G1 C.I. 42040 conjugate,Montmorillonite Basic Red R1 C.I. 45160 conjugate, Montmorillonite C.I.Basic Black 2 conjugate, Hectorite Basic Blue B7 C.I. 42595 conjugate,Hectorite Basic Blue B9 C.I. 52015 conjugate, Hectorite Basic Violet V3C.I. 42555 conjugate, Hectorite Basic Green G1 C.I. 42040 conjugate,Hectorite Basic Red R1 C.I. 45160 conjugate, Hectorite C.I. Basic Black2 conjugate, Saponite Basic Blue B7 C.I. 42595 conjugate, Saponite BasicBlue B9 C.I. 52015 conjugate, Saponite Basic Violet V3 C.I. 42555conjugate, Saponite Basic Green G1 C.I. 42040 conjugate, Saponite BasicRed R1 C.I. 45160 conjugate, Saponite C.I. Basic Black 2 conjugate andmixtures thereof.

Suitable pigments include pigments selected from the group consisting offlavanthrone, indanthrone, chlorinated indanthrone containing from 1 to4 chlorine atoms, pyranthrone, dichloropyranthrone,monobromodichloropyranthrone, dibromodichloropyranthrone,tetrabromopyranthrone, perylene-3,4,9,10-tetracarboxylic acid diimide,wherein the imide groups may be unsubstituted or substituted byC1-C3-alkyl or a phenyl or heterocyclic radical, and wherein the phenyland heterocyclic radicals may additionally carry substituents which donot confer solubility in water, anthrapyrimidinecarboxylic acid amides,violanthrone, isoviolanthrone, dioxazine pigments, copper phthalocyaninewhich may contain up to 2 chlorine atoms per molecule, polychloro-copperphthalocyanine or polybromochloro-copper phthalocyanine containing up to14 bromine atoms per molecule and mixtures thereof. In another aspect,suitable pigments include pigments selected from the group consisting ofUltramarine Blue (C.I. Pigment Blue 29), Ultramarine Violet (C.I.Pigment Violet 15) and mixtures thereof.

The aforementioned fabric hueing agents can be used in combination (anymixture of fabric hueing agents can be used). Suitable fabric hueingagents can be purchased from Aldrich, Milwaukee, Wis., USA; CibaSpecialty Chemicals, Basel, Switzerland; BASF, Ludwigshafen, Germany;Dayglo Color Corporation, Mumbai, India; Organic Dyestuffs Corp., EastProvidence, R.I., USA; Dystar, Frankfurt, Germany; Lanxess, Leverkusen,Germany; Megazyme, Wicklow, Ireland; Clariant, Muttenz, Switzerland;Avecia, Manchester, UK and/or made in accordance with the examplescontained herein.

Suitable hueing agents are described in more detail in U.S. Pat. No.7,208,459 B2.

Encapsulates

In one aspect, an encapsulate comprising a core, a shell having an innerand outer surface, said shell encapsulating said core.

In one aspect of said encapsulate, said core may comprise a materialselected from the group consisting of perfumes; brighteners; dyes;insect repellants; silicones; waxes; flavors; vitamins; fabric softeningagents; skin care agents in one aspect, paraffins; enzymes;anti-bacterial agents; bleaches; sensates; and mixtures thereof; andsaid shell may comprise a material selected from the group consisting ofpolyethylenes; polyamides; polystyrenes; polyisoprenes; polycarbonates;polyesters; polyacrylates; aminoplasts, in one aspect said aminoplastmay comprise a polyureas, polyurethane, and/or polyureaurethane, in oneaspect said polyurea may comprise polyoxymethyleneurea and/or melamineformaldehyde; polyolefins; polysaccharides, in one aspect saidpolysaccharide may comprise alginate and/or chitosan; gelatin; shellac;epoxy resins; vinyl polymers; water insoluble inorganics; silicone; andmixtures thereof.

In one aspect of said encapsulate, said core may comprise perfume.

In one aspect of said encapsulate, said shell may comprise melamineformaldehyde and/or cross linked melamine formaldehyde.

In a one aspect, suitable encapsulates may comprise a core material anda shell, said shell at least partially surrounding said core material,is disclosed. At least 75%, 85% or even 90% of said encapsulates mayhave a fracture strength of from about 0.2 MPa to about 10 MPa, fromabout 0.4 MPa to about 5 MPa, from about 0.6 MPa to about 3.5 MPa, oreven from about 0.7 MPa to about 3 MPa; and a benefit agent leakage offrom 0% to about 30%, from 0% to about 20%, or even from 0% to about 5%.

In one aspect, at least 75%, 85% or even 90% of said encapsulates mayhave a particle size of from about 1 microns to about 80 microns, about5 microns to 60 microns, from about 10 microns to about 50 microns, oreven from about 15 microns to about 40 microns.

In one aspect, at least 75%, 85% or even 90% of said encapsulates mayhave a particle wall thickness of from about 30 nm to about 250 nm, fromabout 80 nm to about 180 nm, or even from about 100 nm to about 160 nm.

In one aspect, said encapsulates' core material may comprise a materialselected from the group consisting of a perfume raw material and/oroptionally a material selected from the group consisting of vegetableoil, including neat and/or blended vegetable oils including caster oil,coconut oil, cottonseed oil, grape oil, rapeseed, soybean oil, corn oil,palm oil, linseed oil, safflower oil, olive oil, peanut oil, coconutoil, palm kernel oil, castor oil, lemon oil and mixtures thereof; estersof vegetable oils, esters, including dibutyl adipate, dibutyl phthalate,butyl benzyl adipate, benzyl octyl adipate, tricresyl phosphate,trioctyl phosphate and mixtures thereof; straight or branched chainhydrocarbons, including those straight or branched chain hydrocarbonshaving a boiling point of greater than about 80° C.; partiallyhydrogenated terphenyls, dialkyl phthalates, alkyl biphenyls, includingmonoisopropylbiphenyl, alkylated naphthalene, includingdipropylnaphthalene, petroleum spirits, including kerosene, mineral oiland mixtures thereof; aromatic solvents, including benzene, toluene andmixtures thereof; silicone oils; and mixtures there of.

In one aspect, said encapsulates' wall material may comprise a suitableresin including the reaction product of an aldehyde and an amine,suitable aldehydes include, formaldehyde. Suitable amines includemelamine, urea, benzoguanamine, glycoluril, and mixtures thereof.Suitable melamines include, methylol melamine, methylated methylolmelamine, imino melamine and mixtures thereof. Suitable ureas include,dimethylol urea, methylated dimethylol urea, urea-resorcinol, andmixtures thereof.

In one aspect, suitable formaldehyde scavengers may be employed with theencapsulates, for example, in a capsule slurry and/or added to a fabricand home care product before, during or after the encapsulates are addedto such fabric and home care product.

Suitable capsules that can be made by following the teaching of USPA2008/0305982 A1; and/or USPA 2009/0247449 A1. Alternatively, suitablecapsules can be purchased from Appleton Papers Inc. of Appleton, Wis.USA.

In addition, the materials for making the aforementioned encapsulatescan be obtained from Solutia Inc. (St. Louis, Mo. U.S.A.), CytecIndustries (West Paterson, N.J. U.S.A.), sigma-Aldrich (St. Louis, Mo.U.S.A.), CP Kelco Corp. of San Diego, Calif., USA; BASF AG ofLudwigshafen, Germany; Rhodia Corp. of Cranbury, N.J., USA; HerculesCorp. of Wilmington, Del., USA; Agrium Inc. of Calgary, Alberta, Canada,ISP of N.J. U.S.A., Akzo Nobel of Chicago, Ill., USA; Stroever ShellacBremen of Bremen, Germany; Dow Chemical Company of Midland, Mich., USA;Bayer AG of Leverkusen, Germany; Sigma-Aldrich Corp., St. Louis, Mo.,USA.

Amphiphilic Cleaning Polymers

Preferably, the amphiphilic cleaning polymer is a compound having thefollowing general structure:bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)-bis((C₂H₅O)(C₂H₄O)n),wherein n=from 20 to 30, and x=from 3 to 8, or sulphated or sulphonatedvariants thereof.

Amphiphilic alkoxylated grease cleaning polymers of the presentinvention refer to any alkoxylated polymers having balanced hydrophilicand hydrophobic properties such that they remove grease particles fromfabrics and surfaces. Specific embodiments of the amphiphilicalkoxylated grease cleaning polymers of the present invention comprise acore structure and a plurality of alkoxylate groups attached to thatcore structure.

The core structure may comprise a polyalkylenimine structure comprising,in condensed form, repeating units of formulae (I), (II), (III) and(IV):

wherein # in each case denotes one-half of a bond between a nitrogenatom and the free binding position of a group A¹ of two adjacentrepeating units of formulae (I), (II), (III) or (IV); * in each casedenotes one-half of a bond to one of the alkoxylate groups; and A¹ isindependently selected from linear or branched C₂-C₆-alkylene; whereinthe polyalkylenimine structure consists of 1 repeating unit of formula(I), x repeating units of formula (II), y repeating units of formula(III) and y+1 repeating units of formula (IV), wherein x and y in eachcase have a value in the range of from 0 to about 150; where the averageweight average molecular weight, Mw, of the polyalkylenimine corestructure is a value in the range of from about 60 to about 10,000g/mol.

The core structure may alternatively comprise a polyalkanolaminestructure of the condensation products of at least one compound selectedfrom N-(hydroxyalkyl)amines of formulae (I.a) and/or (I.b),

wherein A are independently selected from C₁-C₆-alkylene; R¹, R¹*, R²,R²*, R³, R³*, R⁴, R⁴*, R⁵ and R⁵* are independently selected fromhydrogen, alkyl, cycloalkyl or aryl, wherein the last three mentionedradicals may be optionally substituted; and R⁶ is selected fromhydrogen, alkyl, cycloalkyl or aryl, wherein the last three mentionedradicals may be optionally substituted.

The plurality of alkylenoxy groups attached to the core structure areindependently selected from alkylenoxy units of the formula (V)*

A²-O

_(m)

CH₂—CH₂—O

_(n)

A³-O

_(p)R  (V)wherein * in each case denotes one-half of a bond to the nitrogen atomof the repeating unit of formula (I), (II) or (IV); A² is in each caseindependently selected from 1,2-propylene, 1,2-butylene and1,2-isobutylene; A³ is 1,2-propylene; R is in each case independentlyselected from hydrogen and C₁-C₄-alkyl; m has an average value in therange of from 0 to about 2; n has an average value in the range of fromabout 20 to about 50; and p has an average value in the range of fromabout 10 to about 50.

Specific embodiments of the amphiphilic alkoxylated grease cleaningpolymers may be selected from alkoxylated polyalkylenimines having aninner polyethylene oxide block and an outer polypropylene oxide block,the degree of ethoxylation and the degree of propoxylation not goingabove or below specific limiting values. Specific embodiments of thealkoxylated polyalkylenimines according to the present invention have aminimum ratio of polyethylene blocks to polypropylene blocks (n/p) ofabout 0.6 and a maximum of about 1.5(x+2y+1)^(1/2). Alkoxylatedpolyalkyenimines having an n/p ratio of from about 0.8 to about1.2(x+2y+1)^(1/2) have been found to have especially beneficialproperties.

The alkoxylated polyalkylenimines according to the present inventionhave a backbone which consists of primary, secondary and tertiary aminenitrogen atoms which are attached to one another by alkylene radicals Aand are randomly arranged. Primary amino moieties which start orterminate the main chain and the side chains of the polyalkyleniminebackbone and whose remaining hydrogen atoms are subsequently replaced byalkylenoxy units are referred to as repeating units of formulae (I) or(IV), respectively. Secondary amino moieties whose remaining hydrogenatom is subsequently replaced by alkylenoxy units are referred to asrepeating units of formula (II). Tertiary amino moieties which branchthe main chain and the side chains are referred to as repeating units offormula (III).

Since cyclization can occur in the formation of the polyalkyleniminebackbone, it is also possible for cyclic amino moieties to be present toa small extent in the backbone. Such polyalkylenimines containing cyclicamino moieties are of course alkoxylated in the same way as thoseconsisting of the noncyclic primary and secondary amino moieties.

The polyalkylenimine backbone consisting of the nitrogen atoms and thegroups A¹, has an average molecular weight Mw of from about 60 to about10,000 g/mole, preferably from about 100 to about 8,000 g/mole and morepreferably from about 500 to about 6,000 g/mole.

The sum (x+2y+1) corresponds to the total number of alkylenimine unitspresent in one individual polyalkylenimine backbone and thus is directlyrelated to the molecular weight of the polyalkylenimine backbone. Thevalues given in the specification however relate to the number averageof all polyalkylenimines present in the mixture. The sum (x+2y+2)corresponds to the total number amino groups present in one individualpolyalkylenimine backbone.

The radicals A¹ connecting the amino nitrogen atoms may be identical ordifferent, linear or branched C₂-C₆-alkylene radicals, such as1,2-ethylene, 1,2-propylene, 1,2-butylene, 1,2-isobutylene,1,2-pentanediyl, 1,2-hexanediyl or hexamethylen. A preferred branchedalkylene is 1,2-propylene. Preferred linear alkylene are ethylene andhexamethylene. A more preferred alkylene is 1,2-ethylene.

The hydrogen atoms of the primary and secondary amino groups of thepolyalkylenimine backbone are replaced by alkylenoxy units of theformula (V).*

A²-O

_(m)

CH₂—CH₂—O

_(n)

A³O

_(p)—R  (V)

In this formula, the variables preferably have one of the meanings givenbelow:

A² in each case is selected from 1,2-propylene, 1,2-butylene and1,2-isobutylene;

preferably A² is 1,2-propylene. A³ is 1,2-propylene; R in each case isselected from hydrogen and C₁-C₄-alkyl, such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl and tert.-butyl; preferably R is hydrogen.The index m in each case has a value of 0 to about 2; preferably m is 0or approximately 1; more preferably m is 0. The index n has an averagevalue in the range of from about 20 to about 50, preferably in the rangeof from about 22 to about 40, and more preferably in the range of fromabout 24 to about 30. The index p has an average value in the range offrom about 10 to about 50, preferably in the range of from about 11 toabout 40, and more preferably in the range of from about 12 to about 30.

Preferably the alkylenoxy unit of formula (V) is a non-random sequenceof alkoxylate blocks. By non-random sequence it is meant that the[-A²-O—]_(m) is added first (i.e., closest to the bond to the nitrogenatom of the repeating unit of formula (I), (II), or (III)), the[—CH₂—CH₂—O—]_(n) is added second, and the [-A³-O—]_(p) is added third.This orientation provides the alkoxylated polyalkylenimine with an innerpolyethylene oxide block and an outer polypropylene oxide block.

The substantial part of these alkylenoxy units of formula (V) is formedby the ethylenoxy units —[CH₂—CH₂—O—]_(n)— and the propylenoxy units—[CH₂—CH₂(CH₃)—O]_(p)—. The alkylenoxy units may additionally also havea small proportion of propylenoxy or butylenoxy units -[A²-O]_(m)—, i.e.the polyalkylenimine backbone saturated with hydrogen atoms may bereacted initially with small amounts of up to about 2 mol, especiallyfrom about 0.5 to about 1.5 mol, in particular from about 0.8 to about1.2 mol, of propylene oxide or butylene oxide per mole of NH— moietiespresent, i.e. incipiently alkoxylated.

This initial modification of the polyalkylenimine backbone allows, ifnecessary, the viscosity of the reaction mixture in the alkoxylation tobe lowered. However, the modification generally does not influence theperformance properties of the alkoxylated polyalkylenimine and thereforedoes not constitute a preferred measure.

The amphiphilic alkoxylated grease cleaning polymers are present in thefabric and home care products, including but not limited to detergents,of the present invention at levels ranging from about 0.05% to 10% byweight of the fabric and home care product. Embodiments of the fabricand home care products may comprise from about 0.1% to about 5% byweight. More specifically, the embodiments may comprise from about 0.25to about 2.5% of the grease cleaning polymer.

Non-Immunoequivalent Enzyme and/or Additional Enzymes

The fabric and home care products can comprise one or more enzymes whichprovide cleaning performance and/or fabric care benefits. Examples ofsuitable enzymes include, but are not limited to, hemicellulases,peroxidases, proteases, cellulases, xylanases, lipases, phospholipases,esterases, cutinases, pectinases, mannanases, pectate lyases,keratinases, reductases, oxidases, phenoloxidases, lipoxygenases,ligninases, pullulanases, tannases, pentosanases, malanases,ß-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase,and amylases, or mixtures thereof. A typical combination is an enzymecocktail that may comprise, for example, a protease and lipase inconjunction with amylase. When present in a fabric and home careproduct, the aforementioned additional enzymes may be present at levelsfrom about 0.00001% to about 2%, from about 0.0001% to about 1% or evenfrom about 0.001% to about 0.5% enzyme protein by weight of the fabricand home care product.

In one aspect preferred enzymes would include a protease. Suitableproteases include metalloproteases and serine proteases, includingneutral or alkaline microbial serine proteases, such as subtilisins (EC3.4.21.62). Suitable proteases include those of animal, vegetable ormicrobial origin. In one aspect, such suitable protease may be ofmicrobial origin. The suitable proteases include chemically orgenetically modified mutants of the aforementioned suitable proteases.In one aspect, the suitable protease may be a serine protease, such asan alkaline microbial protease or/and a trypsin-type protease. Examplesof suitable neutral or alkaline proteases include:

(a) subtilisins (EC 3.4.21.62), including those derived from Bacillus,such as Bacillus lentus, B. alkalophilus, B. subtilis, B.amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described inU.S. Pat. No. 6,312,936 B1, U.S. Pat. Nos. 5,679,630, 4,760,025,7,262,042 and WO09/021867.

(b) trypsin-type or chymotrypsin-type proteases, such as trypsin (e.g.,of porcine or bovine origin), including the Fusarium protease describedin WO 89/06270 and the chymotrypsin proteases derived from Cellumonasdescribed in WO 05/052161 and WO 05/052146.

(c) metalloproteases, including those derived from Bacillusamyloliquefaciens described in WO 07/044993A2.

Preferred proteases include those derived from Bacillus gibsonii orBacillus Lentus.

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Savinase®, Primase®, Durazym®,Polarzyme®, Kannase®, Liquanase®, Liquanase Ultra®, Savinase Ultra®,Ovozyme®, Neutrase®, Everlase® and Esperase® by Novozymes A/S (Denmark),those sold under the tradename Maxatase®, Maxacal®, Maxapem®,Properase®, Purafect®, Purafect Prime®, Purafect Ox®, FN3®, FN4®,Excellase® and Purafect OXP® by Genencor International, those sold underthe tradename Opticlean® and Optimase® by Solvay Enzymes, thoseavailable from Henkel/ Kemira, namely BLAP (sequence shown in FIG. 29 ofU.S. Pat. No. 5,352,604 with the following mutations S99D+S101R+S103A+V104I+G159S, hereinafter referred to as BLAP), BLAP R (BLAP withS3T+V4I+V199M+V205I+L217D), BLAP X (BLAP with S3T+V4I+V205I) and BLAPF49 (BLAP with S3T+V4I+A194P+V199M+V205I+L217D)—all from Henkel/Kemira;and KAP (Bacillus alkalophilus subtilisin with mutationsA230V+S256G+S259N) from Kao.

In one aspect, the fabric and home care product may comprise a proteasethat is not immunoequivalent to the cold water protease of thisinvention. For the purposes of this invention, an immunoequivalentprotease will have a high degree of identity (>80%) with BPN′ and willcross-react with the same antibody. Suitable non-immunoequivalentenzymes will include those derived from Bacillus Lentus, Bacillusgibsonii and the metalloprotease derived from Bacillusamyloliquefaciens.

Suitable alpha-amylases include those of bacterial or fungal origin.Chemically or genetically modified mutants (variants) are included. Apreferred alkaline alpha-amylase is derived from a strain of Bacillus,such as Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillusstearothermophilus, Bacillus subtilis, or other Bacillus sp., such asBacillus sp. NCIB 12289, NCIB 12512, NCIB 12513, DSM 9375 (U.S. Pat. No.7,153,818) DSM 12368, DSMZ no. 12649, KSM AP1378 (WO 97/00324), KSM K36or KSM K38 (EP 1,022,334). Preferred amylases include:

(a) the variants described in WO 94/02597, WO 94/18314, WO96/23874 andWO 97/43424, especially the variants with substitutions in one or moreof the following positions versus the enzyme listed as SEQ ID No. 2 inWO 96/23874: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190,197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.

(b) the variants described in U.S. Pat. No. 5,856,164 and WO99/23211, WO96/23873, WO00/60060 and WO 06/002643, especially the variants with oneor more substitutions in the following positions versus the AA560 enzymelisted as SEQ ID No. 12 in WO 06/002643:

26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186,193, 203, 214, 231, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298,299, 303, 304, 305, 311, 314, 315, 318, 319, 339, 345, 361, 378, 383,419, 421, 437, 441, 444, 445, 446, 447, 450, 461, 471, 482, 484,preferably that also contain the deletions of D183* and G184*.

(c) variants exhibiting at least 90% identity with SEQ ID No. 4 inWO06/002643, the wild-type enzyme from Bacillus SP722, especiallyvariants with deletions in the 183 and 184 positions and variantsdescribed in WO 00/60060, which is incorporated herein by reference.

(d) variants exhibiting at least 95% identity with the wild-type enzymefrom Bacillus sp. 707 (SEQ ID NO:7 in U.S. Pat. No. 6,093,562),especially those comprising one or more of the following mutations M202,M208, 5255, R172, and/or M261. Preferably said amylase comprises one ormore of M202L, M202V, M202S, M202T, M202I, M202Q, M202W, S255N and/orR172Q. Particularly preferred are those comprising the M202L or M202Tmutations.

Suitable commercially available alpha-amylases include DURAMYL®,LIQUEZYME®, TERMAMYL®, TERMAMYL ULTRA®, NATALASE®, SUPRAMYL®,STAINZYME®, STAINZYME PLUS®, FUNGAMYL® and BAN® (Novozymes A/S,Bagsvaerd, Denmark), KEMZYM® AT 9000 Biozym Biotech Trading GmbHWehlistrasse 27b A-1200 Wien Austria, RAPIDASE®, PURASTAR®, ENZYSIZE®,OPTISIZE HT PLUS® and PURASTAR OXAM® (Genencor International Inc., PaloAlto, Calif.) and KAM® (Kao, 14-10 Nihonbashi Kayabacho, 1-chome,Chuo-ku Tokyo 103-8210, Japan). In one aspect, suitable amylases includeNATALASE®, STAINZYME® and STAINZYME PLUS® and mixtures thereof.

In one aspect, such additional enzyme may be selected from the groupconsisting of: lipases, including “first cycle lipases” such as thosedescribed in U.S. Pat. No. 6,939,702 B1 and US PA 2009/0217464. In oneaspect, the lipase is a first-wash lipase, preferably a variant of thewild-type lipase from Thermomyces lanuginosus comprising T231R and N233Rmutations. The wild-type sequence is the 269 amino acids (amino acids23-291) of the Swissprot accession number Swiss-Prot 059952 (derivedfrom Thermomyces lanuginosus (Humicola lanuginosa)). Preferred lipaseswould include those sold under the tradenames Lipex® and Lipolex®.

In one aspect, other preferred enzymes include microbial-derivedendoglucanases exhibiting endo-beta-1,4-glucanase activity (E.C.3.2.1.4), including a bacterial polypeptide endogenous to a member ofthe genus Bacillus which has a sequence of at least 90%, 94%, 97% andeven 99% identity to the amino acid sequence SEQ ID NO:2 in U.S. Pat.No. 7,141,403B2) and mixtures thereof. Suitable endoglucanases are soldunder the tradenames Celluclean® and Whitezyme® (Novozymes A/S,Bagsvaerd, Denmark).

Other preferred enzymes include pectate lyases sold under the tradenamesPectawash®, Pectaway® and mannanases sold under the tradenames Mannaway®(all from Novozymes A/S, Bagsvaerd, Denmark), and Purabrite® (GenencorInternational Inc., Palo Alto, Calif.).

Adjunct Materials

While not essential for the purposes of the present invention, thenon-limiting list of adjuncts illustrated hereinafter are suitable foruse in the instant fabric and home care products and may be desirablyincorporated in certain embodiments of the invention, for example toassist or enhance cleaning performance, for treatment of the substrateto be cleaned, or to modify the aesthetics of the fabric and home careproduct as is the case with perfumes, colorants, dyes or the like. Thelevels of any such adjuncts incorporated in any fabric and home careproduct are in addition to any materials previously recited forincorporation. The precise nature of these additional components, andlevels of incorporation thereof, will depend on the physical form of thefabric and home care product and the nature of the cleaning operationfor which it is to be used. Suitable adjunct materials include, but arenot limited to, surfactants, builders, chelating agents, dye transferinhibiting agents, dispersants, additional enzymes, and enzymestabilizers, catalytic materials, bleach activators, hydrogen peroxide,sources of hydrogen peroxide, preformed peracids, polymeric dispersingagents, clay soil removal/anti-redeposition agents, brighteners, sudssuppressors, dyes, perfumes, perfume delivery systems, structureelasticizing agents, fabric softeners, carriers, hydrotropes, processingaids, solvents and/or pigments. In addition to the disclosure below,suitable examples of such other adjuncts and levels of use are found inU.S. Pat. Nos. 5,576,282, 6,306,812 B1 and 6,326,348 B1 that areincorporated by reference.

As stated, the adjunct ingredients are not essential to Applicants'fabric and home care products. Thus, certain embodiments of Applicants'fabric and home care products do not contain one or more of thefollowing adjuncts materials: surfactants, builders, chelating agents,dye transfer inhibiting agents, dispersants, additional enzymes, andenzyme stabilizers, catalytic materials, bleach activators, hydrogenperoxide, sources of hydrogen peroxide, preformed peracids, polymericdispersing agents, clay soil removal/anti-redeposition agents,brighteners, suds suppressors, dyes, perfumes, perfume delivery systems,structure elasticizing agents, fabric softeners, carriers, hydrotropes,processing aids, solvents and/or pigments. However, when one or moreadjuncts are present, such one or more adjuncts may be present asdetailed below:

Perfume Delivery Systems and Perfumes—The compositions of the presentinvention may comprise a neat perfume and/or perfume technology systemsthat can be combined to yield the desired scent experience from thestore shelf stage of a product, through its total performance cycle.Suitable perfumes include those perfumes that are enduring perfumesand/or quadrant perfumes. Examples of such neat perfumes are disclosedU.S. Pat. Nos. 5,500,138; 5,500,154; 6,491,728; 5,500,137 and 5,780,404.Suitable perfume delivery systems, methods of making certain perfumedelivery systems and the uses of such perfume delivery systems aredisclosed in USPA 2007/0275866 A1. Such perfume delivery systemsinclude:

-   -   Polymer Assisted Delivery (PAD): This perfume delivery        technology uses polymeric materials to deliver perfume        materials. Classical coacervation, water soluble or partly        soluble to insoluble charged or neutral polymers, liquid        crystals, hot melts, hydrogels, perfumed plastics,        microcapsules, nano- and micro-latexes, polymeric film formers,        and polymeric absorbents, polymeric adsorbents, etc. are some        examples. PAD includes but is not limited to:        -   a.) Matrix Systems: The fragrance is dissolved or dispersed            in a polymer matrix or particle. Perfumes, for example, may            be 1) dispersed into the polymer prior to formulating into            the product or 2) added separately from the polymer during            or after formulation of the product. Diffusion of perfume            from the polymer is a common trigger that allows or            increases the rate of perfume release from a polymeric            matrix system that is deposited or applied to the desired            surface (situs), although many other triggers are know that            may control perfume release. Absorption and/or adsorption            into or onto polymeric particles, films, solutions, and the            like are aspects of this technology. Nano- or            micro-particles composed of organic materials (e.g.,            latexes) are examples. Suitable particles include a wide            range of materials including, but not limited to polyacetal,            polyacrylate, polyacrylic, polyacrylonitrile, polyamide,            polyaryletherketone, polybutadiene, polybutylene,            polybutylene terephthalate, polychloroprene, poly ethylene,            polyethylene terephthalate, polycyclohexylene dimethylene            terephthalate, polycarbonate, polychloroprene,            polyhydroxyalkanoate, polyketone, polyester, polyethylene,            polyetherimide, polyethersulfone, polyethylenechlorinates,            polyimide, polyisoprene, polylactic acid, polymethylpentene,            polyphenylene oxide, polyphenylene sulfide, polyphthalamide,            polypropylene, polystyrene, polysulfone, polyvinyl acetate,            polyvinyl chloride, as well as polymers or copolymers based            on acrylonitrile-butadiene, cellulose acetate,            ethylene-vinyl acetate, ethylene vinyl alcohol,            styrene-butadiene, vinyl acetate-ethylene, and mixtures            thereof.        -   “Standard” systems refer to those that are “pre-loaded” with            the intent of keeping the pre-loaded perfume associated with            the polymer until the moment or moments of perfume release.            Such polymers may also suppress the neat product odor and            provide a bloom and/or longevity benefit depending on the            rate of perfume release. One challenge with such systems is            to achieve the ideal balance between 1) in-product stability            (keeping perfume inside carrier until you need it) and 2)            timely release (during use or from dry situs). Achieving            such stability is particularly important during in-product            storage and product aging. This challenge is particularly            apparent for aqueous-based, surfactant-containing products,            such as heavy duty liquid laundry detergents. Many            “Standard” matrix systems available effectively become            “Equilibrium” systems when formulated into aqueous-based            products. One may select an “Equilibrium” system or a            Reservoir system, which has acceptable in-product diffusion            stability and available triggers for release (e.g.,            friction). “Equilibrium” systems are those in which the            perfume and polymer may be added separately to the product,            and the equilibrium interaction between perfume and polymer            leads to a benefit at one or more consumer touch points            (versus a free perfume control that has no polymer-assisted            delivery technology). The polymer may also be pre-loaded            with perfume; however, part or all of the perfume may            diffuse during in-product storage reaching an equilibrium            that includes having desired perfume raw materials (PRMs)            associated with the polymer. The polymer then carries the            perfume to the surface, and release is typically via perfume            diffusion. The use of such equilibrium system polymers has            the potential to decrease the neat product odor intensity of            the neat product (usually more so in the case of pre-loaded            standard system). Deposition of such polymers may serve to            “flatten” the release profile and provide increased            longevity. As indicated above, such longevity would be            achieved by suppressing the initial intensity and may enable            the formulator to use more high impact or low odor detection            threshold (ODT) or low Kovats Index (KI) PRMs to achieve            FMOT benefits without initial intensity that is too strong            or distorted. It is important that perfume release occurs            within the time frame of the application to impact the            desired consumer touch point or touch points. Suitable            micro-particles and micro-latexes as well as methods of            making same may be found in USPA 2005/0003980 A1. Matrix            systems also include hot melt adhesives and perfume            plastics. In addition, hydrophobically modified            polysaccharides may be formulated into the perfumed product            to increase perfume deposition and/or modify perfume            release. All such matrix systems, including for example            polysaccharides and nanolatexes may be combined with other            PDTs, including other PAD systems such as PAD reservoir            systems in the form of a perfume microcapsule (PMC). Polymer            Assisted Delivery (PAD) matrix systems may include those            described in the following references: US Patent            Applications 2004/0110648 A1; 2004/0092414 A1; 2004/0091445            A1 and 2004/0087476 A1; and U.S. Pat. Nos. 6,531,444;            6,024,943; 6,042,792; 6,051,540; 4,540,721 and 4,973,422.        -   Silicones are also examples of polymers that may be used as            PDT, and can provide perfume benefits in a manner similar to            the polymer-assisted delivery “matrix system”. Such a PDT is            referred to as silicone-assisted delivery (SAD). One may            pre-load silicones with perfume, or use them as an            equilibrium system as described for PAD. Suitable silicones            as well as making same may be found in WO 2005/102261; USPA            20050124530A1; USPA 20050143282A1; and WO 2003/015736.            Functionalized silicones may also be used as described in            USPA 2006/003913 A1. Examples of silicones include            polydimethylsiloxane and polyalkyldimethylsiloxanes. Other            examples include those with amine functionality, which may            be used to provide benefits associated with amine-assisted            delivery (AAD) and/or polymer-assisted delivery (PAD) and/or            amine-reaction products (ARP). Other such examples may be            found in U.S. Pat. No. 4,911,852; USPA 2004/0058845 A1; USPA            2004/0092425 A1 and USPA 2005/0003980 A1.        -   b.) Reservoir Systems: Reservoir systems are also known as a            core-shell type technology, or one in which the fragrance is            surrounded by a perfume release controlling membrane, which            may serve as a protective shell. The material inside the            microcapsule is referred to as the core, internal phase, or            fill, whereas the wall is sometimes called a shell, coating,            or membrane. Microparticles or pressure sensitive capsules            or microcapsules are examples of this technology.            Microcapsules of the current invention are formed by a            variety of procedures that include, but are not limited to,            coating, extrusion, spray-drying, interfacial, in-situ and            matrix polymerization. The possible shell materials vary            widely in their stability toward water. Among the most            stable are polyoxymethyleneurea (PMU)-based materials, which            may hold certain PRMs for even long periods of time in            aqueous solution (or product). Such systems include but are            not limited to urea-formaldehyde and/or            melamine-formaldehyde. Gelatin-based microcapsules may be            prepared so that they dissolve quickly or slowly in water,            depending for example on the degree of cross-linking. Many            other capsule wall materials are available and vary in the            degree of perfume diffusion stability observed. Without            wishing to be bound by theory, the rate of release of            perfume from a capsule, for example, once deposited on a            surface is typically in reverse order of in-product perfume            diffusion stability. As such, urea-formaldehyde and            melamine-formaldehyde microcapsules for example, typically            require a release mechanism other than, or in addition to,            diffusion for release, such as mechanical force (e.g.,            friction, pressure, shear stress) that serves to break the            capsule and increase the rate of perfume (fragrance)            release. Other triggers include melting, dissolution,            hydrolysis or other chemical reaction, electromagnetic            radiation, and the like. The use of pre-loaded microcapsules            requires the proper ratio of in-product stability and in-use            and/or on-surface (on-situs) release, as well as proper            selection of PRMs. Microcapsules that are based on            urea-formaldehyde and/or melamine-formaldehyde are            relatively stable, especially in near neutral aqueous-based            solutions. Other suitable microcapsules include            microcapsules having shells that comprise urethane            materials, acrylics and/or vinyl alcohols. These materials            may require a friction trigger which may not be applicable            to all product applications. Other microcapsule materials            (e.g., gelatin) may be unstable in aqueous-based products            and may even provide reduced benefit (versus free perfume            control) when in-product aged. Scratch and sniff            technologies are yet another example of PAD. Perfume            microcapsules (PMC) may include those described in the            following references: US Patent Applications: 2003/0125222            A1; 2003/215417 A1; 2003/216488 A1; 2003/158344 A1;            2003/165692 A1; 2004/071742 A1; 2004/071746 A1; 2004/072719            A1; 2004/072720 A1; 2006/0039934 A1; 2003/203829 A1;            2003/195133 A1; 2004/087477 A1; 2004/0106536 A1; and U.S.            Pat. Nos. 6,645,479 B1; 6,200,949 B1; 4,882,220; 4,917,920;            4,514,461; 6,106,875 and 4,234,627, 3,594,328 and U.S. Pat.            No. RE 32713.

-   Molecule-Assisted Delivery (MAD): Non-polymer materials or molecules    may also serve to improve the delivery of perfume. Without wishing    to be bound by theory, perfume may non-covalently interact with    organic materials, resulting in altered deposition and/or release.    Non-limiting examples of such organic materials include but are not    limited to hydrophobic materials such as organic oils, waxes,    mineral oils, petrolatum, fatty acids or esters, sugars,    surfactants, liposomes and even other perfume raw material (perfume    oils), as well as natural oils, including body and/or other soils.    Perfume fixatives are yet another example. In one aspect,    non-polymeric materials or molecules have a CLogP greater than    about 2. Molecule-Assisted Delivery (MAD) may also include those    described in U.S. Pat. Nos. 7,119,060 and 5,506,201.

-   Fiber-Assisted Delivery (FAD): The choice or use of a situs itself    may serve to improve the delivery of perfume. In fact, the situs    itself may be a perfume delivery technology. For example, different    fabric types such as cotton or polyester will have different    properties with respect to ability to attract and/or retain and/or    release perfume. The amount of perfume deposited on or in fibers may    be altered by the choice of fiber, and also by the history or    treatment of the fiber, as well as by any fiber coatings or    treatments. Fibers may be woven and non-woven as well as natural or    synthetic. Natural fibers include those produced by plants, animals,    and geological processes, and include but are not limited to    cellulose materials such as cotton, linen, hemp jute, flax, ramie,    and sisal, and fibers used to manufacture paper and cloth.    Fiber-Assisted Delivery may consist of the use of wood fiber, such    as thermomechanical pulp and bleached or unbleached kraft or sulfite    pulps. Animal fibers consist largely of particular proteins, such as    silk, sinew, catgut and hair (including wool). Polymer fibers based    on synthetic chemicals include but are not limited to polyamide    nylon, PET or PBT polyester, phenol-formaldehyde (PF), polyvinyl    alcohol fiber (PVOH), polyvinyl chloride fiber (PVC), polyolefins    (PP and PE), and acrylic polymers. All such fibers may be pre-loaded    with a perfume, and then added to a product that may or may not    contain free perfume and/or one or more perfume delivery    technologies. In one aspect, the fibers may be added to a product    prior to being loaded with a perfume, and then loaded with a perfume    by adding a perfume that may diffuse into the fiber, to the product.    Without wishing to be bound by theory, the perfume may absorb onto    or be adsorbed into the fiber, for example, during product storage,    and then be released at one or more moments of truth or consumer    touch points.

-   Amine Assisted Delivery (AAD): The amine-assisted delivery    technology approach utilizes materials that contain an amine group    to increase perfume deposition or modify perfume release during    product use. There is no requirement in this approach to pre-complex    or pre-react the perfume raw material(s) and amine prior to addition    to the product. In one aspect, amine-containing AAD materials    suitable for use herein may be non-aromatic; for example,    polyalkylimine, such as polyethyleneimine (PEI), or polyvinylamine    (PVAm), or aromatic, for example, anthranilates. Such materials may    also be polymeric or non-polymeric. In one aspect, such materials    contain at least one primary amine. This technology will allow    increased longevity and controlled release also of low ODT perfume    notes (e.g., aldehydes, ketones, enones) via amine functionality,    and delivery of other PRMs, without being bound by theory, via    polymer-assisted delivery for polymeric amines Without technology,    volatile top notes can be lost too quickly, leaving a higher ratio    of middle and base notes to top notes. The use of a polymeric amine    allows higher levels of top notes and other PRMS to be used to    obtain freshness longevity without causing neat product odor to be    more intense than desired, or allows top notes and other PRMs to be    used more efficiently. In one aspect, AAD systems are effective at    delivering PRMs at pH greater than about neutral. Without wishing to    be bound by theory, conditions in which more of the amines of the    AAD system are deprotonated may result in an increased affinity of    the deprotonated amines for PRMs such as aldehydes and ketones,    including unsaturated ketones and enones such as damascone. In    another aspect, polymeric amines are effective at delivering PRMs at    pH less than about neutral. Without wishing to be bound by theory,    conditions in which more of the amines of the AAD system are    protonated may result in a decreased affinity of the protonated    amines for PRMs such as aldehydes and ketones, and a strong affinity    of the polymer framework for a broad range of PRMs. In such an    aspect, polymer-assisted delivery may be delivering more of the    perfume benefit; such systems are a subspecies of AAD and may be    referred to as Amine-Polymer-Assisted Delivery or APAD. In some    cases when the APAD is employed in a composition that has a pH of    less than seven, such APAD systems may also be considered    Polymer-Assisted Delivery (PAD). In yet another aspect, AAD and PAD    systems may interact with other materials, such as anionic    surfactants or polymers to form coacervate and/or coacervates-like    systems. In another aspect, a material that contains a heteroatom    other than nitrogen, for example sulfur, phosphorus or selenium, may    be used as an alternative to amine compounds. In yet another aspect,    the aforementioned alternative compounds can be used in combination    with amine compounds. In yet another aspect, a single molecule may    comprise an amine moiety and one or more of the alternative    heteroatom moieties, for example, thiols, phosphines and selenols.    Suitable AAD systems as well as methods of making same may be found    in US Patent Applications 2005/0003980 A1; 2003/0199422 A1;    2003/0036489 A1; 2004/0220074 A1 and U.S. Pat. No. 6,103,678.

-   Cyclodextrin Delivery System (CD): This technology approach uses a    cyclic oligosaccharide or cyclodextrin to improve the delivery of    perfume. Typically a perfume and cyclodextrin (CD) complex is    formed. Such complexes may be preformed, formed in-situ, or formed    on or in the situs. Without wishing to be bound by theory, loss of    water may serve to shift the equilibrium toward the CD-Perfume    complex, especially if other adjunct ingredients (e.g., surfactant)    are not present at high concentration to compete with the perfume    for the cyclodextrin cavity. A bloom benefit may be achieved if    water exposure or an increase in moisture content occurs at a later    time point. In addition, cyclodextrin allows the perfume formulator    increased flexibility in selection of PRMs. Cyclodextrin may be    pre-loaded with perfume or added separately from perfume to obtain    the desired perfume stability, deposition or release benefit.    Suitable CDs as well as methods of making same may be found in USPA    2005/0003980 A1 and 2006/0263313 A1 and U.S. Pat. Nos. 5,552,378;    3,812,011; 4,317,881; 4,418,144 and 4,378,923.

-   Starch Encapsulated Accord (SEA): The use of a starch encapsulated    accord (SEA) technology allows one to modify the properties of the    perfume, for example, by converting a liquid perfume into a solid by    adding ingredients such as starch. The benefit includes increased    perfume retention during product storage, especially under    non-aqueous conditions. Upon exposure to moisture, a perfume bloom    may be triggered. Benefits at other moments of truth may also be    achieved because the starch allows the product formulator to select    PRMs or PRM concentrations that normally cannot be used without the    presence of SEA. Another technology example includes the use of    other organic and inorganic materials, such as silica to convert    perfume from liquid to solid. Suitable SEAs as well as methods of    making same may be found in USPA 2005/0003980 A1 and U.S. Pat. No.    6,458,754 B1.

In one aspect, SEA's may be made by preparing a mixture comprisingstarch, water, acid and a perfume, the acid being incorporated in themixture in an amount sufficient to lower the pH of the starch-watermixture by at least 0.25 units; and atomising and drying the mixturethereby forming encapsulated perfume. In the first step in the processof perfume encapsulation, an aqueous mixture is prepared comprisingstarch, water, perfume and acid. These ingredients may be added in anyorder, but usually the starch-water mixture is prepared first andsubsequently, either sequentially or together, the acid and perfume areadded. When they are added sequentially, the acid may be added prior tothe ingredient for encapsulation. Alternatively, the acid is added afterthe ingredient for encapsulation. The concentration of starch in theaqueous mixture may be from as low as 5 or 10 wt % to as high as 60 oreven 75 wt %. Generally the concentration of starch in the mixture isfrom 20 to 50 wt %, more usually around 25 to 40 wt % in the aqueousmixture.

-   Suitable starches can be made from raw starch, pregelatinized    starch, modified starch derived from tubers, legumes, cereal and    grains for example corn starch, wheat starch, rice starch, waxy corn    starch, oat starch, cassava starch, waxy barley starch, waxy rice    starch, sweet rice starch, amioca, potato starch, tapioca starch and    mixtures thereof. Modified starches may be particularly suitable for    use in the present invention, and these include hydrolyzed starch,    acid thinned starch, starch having hydrophobic groups, such as    starch esters of long chain hydrocarbons (C5 or greater), starch    acetates, starch octenyl succinate and mixtures thereof. In one    aspect, starch esters, such as starch octenyl succinates are    employed.-   The term “hydrolyzed starch” refers to oligosaccharide-type    materials that are typically obtained by acid and/or enzymatic    hydrolysis of starches, preferably corn starch. It may be preferred    to include in the starch water-mixture, a starch ester. Particularly    preferred are the modified starches comprising a starch derivative    containing a hydrophobic group or both a hydrophobic and a    hydrophilic group which has been degraded by at least one enzyme    capable of cleaving the 1,4 linkages of the starch molecule from the    non-reducing ends to produce short chained saccharides to provide    high oxidation resistance while maintaining substantially high    molecular weight portions of the starch base. The aqueous starch    mixture may also include a plasticizer for the starch. Suitable    examples include monosaccharides, disaccharides, oligosaccharides    and maltodextrins, such as glucose, sucrose, sorbitol, gum arabic,    guar gums and maltodextrin.-   The acid used in the process of the invention may be any acid.    Examples include sulphuric acid, nitric acid, hydrochloric acid,    sulphamic acid and phosphonic acid. In one aspect, carboxylic    organic acids are employed. In another aspect, organic acids    comprising more than one carboxylic acid groups are employed.    Examples of suitable organic acids include citric acid, tartaric    acid, maleic acid, malic acid, succinic acid, sebacic acid, adipic    acid, itaconic acid, acetic acid and ascorbic acid, etc. In one    aspect, saturated acids, such as citric acid, are employed.-   Suitable perfumes for encapsulation include the HIA perfumes    including those having a boiling point determined at the normal    standard pressure of about 760 mmHg of 275° C. or lower, an    octanol/water partition coefficient P of about 2000 or higher and an    odour detection threshold of less than or equal 50 parts per billion    (ppb). In one aspect, the perfume may have log P of 2 or higher.    Suitable perfumes may be selected from the group consisting of    3-(4-t-butylphenyl)-2-methyl propanal, 3-(4-t-butylphenyl)-propanal,    3-(4-isopropylphenyl)-2-methylpropanal,    methylenedioxyphenyl)-2-methylpropanal, and 2,6-dimethyl-5-heptenal,    Alpha-damascone, Delta-damascone, Iso-damascone, Beta-damascenone,    6,7-dihydro-1,1,2,3,3-pentamethyl-4(5H)-indanone,    methyl-7,3-dihydro-2H-1,5-benzodioxepine-3-one,    2-[2-(4-methyl-3-cyclohexenyl-1-yl)propyl]cyclopentan-2-one,    2-sec-butylcyclohexanone, and Alpha-dihydro ionone, linalool,    ethyllinalool, tetrahydrolinalool, and dihydromyrcenol.-   Suitable ingredients can be obtained from Givaudan of Mount Olive,    N.J., USA, International Flavors & Fragrances of South Brunswick,    N.J., USA, or Quest of Naarden, Netherlands.-   Following the formation of the aqueous mixture comprising starch,    water, perfumes and acid, the mixture is mixed under high shear to    form an emulsion or dispersion of ingredient for encapsulation in    the aqueous starch solution.-   Any suitable technique may then be used for the final stage of    processing where the aqueous mixture including acid and perfumes is    atomised and dried. Suitable techniques include, but are not limited    to those known in the art including spray drying, extrusion, spray    chilling/crystallisation methods, fluid bed coating and the use of    phase transfer catalysts to promote interfacial polymerization.    Spray efficiencies may be increased by methods known in the art,    such as by using high drying towers, lightly oiling the chamber    walls, or using preconditioned air in which the moisture has been    substantially removed.-   Inorganic Carrier Delivery System (ZIC): This technology relates to    the use of porous zeolites or other inorganic materials to deliver    perfumes. Perfume-loaded zeolite may be used with or without adjunct    ingredients used for example to coat the perfume-loaded zeolite    (PLZ) to change its perfume release properties during product    storage or during use or from the dry situs. Suitable zeolite and    inorganic carriers as well as methods of making same may be found in    USPA 2005/0003980 A1 and U.S. Pat. Nos. 5,858,959; 6,245,732 B1;    6,048,830 and 4,539,135. Silica is another form of ZIC. Another    example of a suitable inorganic carrier includes inorganic tubules,    where the perfume or other active material is contained within the    lumen of the nano- or micro-tubules. Preferably, the perfume-loaded    inorganic tubule (or Perfume-Loaded Tubule or PLT) is a mineral    nano- or micro-tubule, such as halloysite or mixtures of halloysite    with other inorganic materials, including other clays. The PLT    technology may also comprise additional ingredients on the inside    and/or outside of the tubule for the purpose of improving in-product    diffusion stability, deposition on the desired situs or for    controlling the release rate of the loaded perfume. Monomeric and/or    polymeric materials, including starch encapsulation, may be used to    coat, plug, cap, or otherwise encapsulate the PLT. Suitable PLT    systems as well as methods of making same may be found in U.S. Pat.    No. 5,651,976.-   Pro-Perfume (PP): This technology refers to perfume technologies    that result from the reaction of perfume materials with other    substrates or chemicals to form materials that have a covalent bond    between one or more PRMs and one or more carriers. The PRM is    converted into a new material called a pro-PRM (i.e., pro-perfume),    which then may release the original PRM upon exposure to a trigger    such as water or light. Pro-perfumes may provide enhanced perfume    delivery properties such as increased perfume deposition, longevity,    stability, retention, and the like. Pro-perfumes include those that    are monomeric (non-polymeric) or polymeric, and may be pre-formed or    may be formed in-situ under equilibrium conditions, such as those    that may be present during in-product storage or on the wet or dry    situs. Nonlimiting examples of pro-perfumes include Michael adducts    (e.g., beta-amino ketones), aromatic or non-aromatic imines (Schiffs    Bases), oxazolidines, beta-keto esters, and orthoesters. Another    aspect includes compounds comprising one or more beta-oxy or    beta-thio carbonyl moieties capable of releasing a PRM, for example,    an alpha, beta-unsaturated ketone, aldehyde or carboxylic ester. The    typical trigger for perfume release is exposure to water; although    other triggers may include enzymes, heat, light, pH change,    autoxidation, a shift of equilibrium, change in concentration or    ionic strength and others. For aqueous-based products,    light-triggered pro-perfumes are particularly suited. Such    photo-pro-perfumes (PPPs) include but are not limited to those that    release coumarin derivatives and perfumes and/or pro-perfumes upon    being triggered. The released pro-perfume may release one or more    PRMs by means of any of the above mentioned triggers. In one aspect,    the photo-pro-perfume releases a nitrogen-based pro-perfume when    exposed to a light and/or moisture trigger. In another aspect, the    nitrogen-based pro-perfume, released from the photo-pro-perfume,    releases one or more PRMs selected, for example, from aldehydes,    ketones (including enones) and alcohols. In still another aspect,    the PPP releases a dihydroxy coumarin derivative. The    light-triggered pro-perfume may also be an ester that releases a    coumarin derivative and a perfume alcohol. In one aspect the    pro-perfume is a dimethoxybenzoin derivative as described in USPA    2006/0020459 A1. In another aspect the pro-perfume is a 3′,    5′-dimethoxybenzoin (DMB) derivative that releases an alcohol upon    exposure to electromagnetic radiation. In yet another aspect, the    pro-perfume releases one or more low ODT PRMs, including tertiary    alcohols such as linalool, tetrahydrolinalool, or dihydromyrcenol.    Suitable pro-perfumes and methods of making same can be found in    U.S. Pat. Nos. 7,018,978 B2; 6,987,084 B2; 6,956,013 B2; 6,861,402    B1; 6,544,945 B1; 6,093,691; 6,277,796 B1; 6,165,953; 6,316,397 B1;    6,437,150 B1; 6,479,682 B1; 6,096,918; 6,218,355 B1; 6,133,228;    6,147,037; 7,109,153 B2; 7,071,151 B2; 6,987,084 B2; 6,610,646 B2    and 5,958,870, as well as can be found in USPA 2005/0003980 A1 and    USPA 2006/0223726 A1.    -   a.) Amine Reaction Product (ARP): For purposes of the present        application, ARP is a subclass or species of PP. One may also        use “reactive” polymeric amines in which the amine functionality        is pre-reacted with one or more PRMs to form an amine reaction        product (ARP). Typically the reactive amines are primary and/or        secondary amines, and may be part of a polymer or a monomer        (non-polymer). Such ARPs may also be mixed with additional PRMs        to provide benefits of polymer-assisted delivery and/or        amine-assisted delivery. Nonlimiting examples of polymeric        amines include polymers based on polyalkylimines, such as        polyethyleneimine (PEI), or polyvinylamine (PVAm). Nonlimiting        examples of monomeric (non-polymeric) amines include hydroxyl        amines, such as 2-aminoethanol and its alkyl substituted        derivatives, and aromatic amines such as anthranilates. The ARPs        may be premixed with perfume or added separately in leave-on or        rinse-off applications. In another aspect, a material that        contains a heteroatom other than nitrogen, for example oxygen,        sulfur, phosphorus or selenium, may be used as an alternative to        amine compounds. In yet another aspect, the aforementioned        alternative compounds can be used in combination with amine        compounds. In yet another aspect, a single molecule may comprise        an amine moiety and one or more of the alternative heteroatom        moieties, for example, thiols, phosphines and selenols. The        benefit may include improved delivery of perfume as well as        controlled perfume release. Suitable ARPs as well as methods of        making same can be found in USPA 2005/0003980 A1 and U.S. Pat.        No. 6,413,920 B1.

In one aspect, the amine reaction product's perfume component, which isreacted with the amine to form the amine reaction product, is selectedfrom a perfume comprising a ketone moiety and/or an aldehyde moiety. Inone aspect, such perfumes comprise a chain containing at least 5 carbonatoms. In one aspect, suitable perfumes comprising a ketone moiety maybe selected from Alpha Damascone, Delta Damascone, Iso Damascone,Carvone, Gamma-Methyl-Ionone, Iso-E-Super,2,4,4,7-Tetramethyl-oct-6-en-3-one, Benzyl Acetone, Beta Damascone,Damascenone, methyl dihydrojasmonate, methyl cedrylone, and mixturesthereof. In one aspect, suitable perfumes comprising an aldehyde moietymay be selected from 1-decanal, benzaldehyde, florhydral,2,4-dimethyl-3-cyclohexen-1-carboxaldehyde;cis/trans-3,7-dimethyl-2,6-octadien-1-al; heliotropin;2,4,6-trimethyl-3-cyclohexene-1-carboxaldehyde; 2,6-nonadienal;alpha-n-amyl cinnamic aldehyde, alpha-n-hexyl cinnamic aldehyde, P.T.Bucinal, lyral, cymal, methyl nonyl acetaldehyde, hexanal,trans-2-hexenal, and mixture thereof. In one aspect, the suitableperfume mat be selected from undecylenic aldehyde, undecalactone gamma,heliotropin, dodecalactone gamma, p-anisic aldehyde, parahydroxy-phenyl-butanone, cymal, benzyl acetone, ionone alpha,p.t.bucinal, damascenone, ionone beta and methyl-nonyl ketone, and/ormixtures thereof. Typically the level of perfume may be from 10% to 90%,from 30% to 85%, or even from 45% to 80% by weight of the amine reactionproduct. In one aspect, suitable amine reaction products are thoseresulting from the reaction of polethyleneimine polymer like Lupasolpolymers, with one or more of the following Alpha Damascone, DeltaDamascone, Carvone, Hedione, Florhydral, Lilial, Heliotropine,Gamma-Methyl-Ionone and 2,4-dimethyl-3-cyclohexen-1-carboxaldehyde;amine reaction products are those resulting from the reaction ofAstramol Dendrimers with Carvone and amine reaction products resultingfrom the reaction of ethyl-4-amino benzoate with2,4-dimethyl-3-cyclohexen-l-carboxaldehyde. In one aspect, suitableamine reaction products are those resulting from the reaction of LupasolHF with Delta Damascone; LupasolG35 with Alpha Damascone; LupasolG100with 2,4-dimethyl-3-cyclohexen-1-carboxaldehyde, ethyl-4-amino benzoatewith 2,4-dimethyl-3-cyclohexen-1-carboxaldehyde.

-   In one aspect, suitable primary and/or secondary amine containing    compounds are characterized by an Odor Intensity Index of less than    that of a 1% solution of methylanthranilate in dipropylene glycol.-   A general structure for a suitable primary amine compound is as    follows:    B—(NH₂)_(n)    wherein B is a carrier material, and n is an index of value of at    least 1.-   Suitable compounds comprising a secondary amine group may have a    structure similar to the above excepted that the compound comprises    one or more —NH— moieties in addition to any —NH2 moieties. Thus,    such an amine compound may have the formula:    B—(NH₂)_(n); B—(NH)_(n); B—(NH)_(n)—(NH₂)_(n)    wherein B is a carrier material, and each n is independently an    index of value of at least 1.-   In one aspect, B carriers may be inorganic having non-or    substantially non carbon based backbones, or organic carriers having    essentially carbon bond backbones.-   Suitable inorganic carriers include mono or polymers or    organic-organosilicon copolymers of amino derivatised organo silane,    siloxane, silazane, alumane, aluminum siloxane, or aluminum silicate    compounds. Typical examples of such carriers are: organosiloxanes    with at least one primary amine moiety like the diaminoalkylsiloxane    [H₂NCH₂(CH₃)₂Si]O, or the organoaminosilane (C₆H₅) 3SiNH₂ (described    in: Chemistry and Technology of Silicone, W. Noll, Academic Press    Inc. 1998, London, pp 209, 106). Mono or polymer or    organic-organosilicon copolymers containing one or more    organosilylhydrasine moiety are also suitable. A typical example of    such a carrier material is N,N′-bis(trimethylsilyl)hydrazine    (Me₃Si)₂NNH₂. Typical suitable amines comprising an organic carrier    include aminoaryl derivatives, polyamines, aminoacids and    derivatives, substituted amines and amides, glucamines, dendrimers    and amino-substituted mono-, di-, oligo-, poly-saccharides.-   The amine compound may be interrupted or substituted by linkers or    cellulose substantive group. A general formula for this amine    compound is as follows:    NH_(2n)-L_(m)-B-L_(m)-R*_(m);    wherein each m is an index of value 0 or at least 1, and n is an    index of value of at least 1 as defined herein before. As can be    seen above, the amine group is linked to a carrier molecule as    defined by classes hereinafter described. The primary and/or    secondary amine group is either directly linked to the carrier group    or via a linker group L. The carrier can also be substituted by a R*    substituent, and R* can be linked to the carrier either directly or    via a linker group L. R* can also contain branching groups like e.g.    tertiary amine and amide groups.-   It is important for the purpose of the invention that the amine    compound comprises at least one primary and/or secondary amine group    to react with the perfume aldehyde and/or ketone to form the    reaction products. Such reaction is typically known as a Schiff base    reaction as a Schiff base is formed. The amine compound is not    limited to having only one amine function. Indeed, more preferably,    the amine compound comprises more than one amine function, thereby    enabling the amine compound to react with several aldehydes and/or    ketones. Accordingly, reaction products carrying mixed aldehyde(s)    and/or ketone(s) can be achieved, thereby resulting in a mixed    release of such fragrances.

Bleaching Agents—The fabric and home care products of the presentinvention may comprise one or more bleaching agents. Suitable bleachingagents other than bleaching catalysts include photobleaches, bleachactivators, hydrogen peroxide, sources of hydrogen peroxide, pre-formedperacids and mixtures thereof. In general, when a bleaching agent isused, the fabric and home care products of the present invention maycomprise from about 0.1% to about 50% or even from about 0.1% to about25% bleaching agent by weight of the subject fabric and home careproduct. Examples of suitable bleaching agents include:

(1) photobleaches for example sulfonated zinc phthalocyanine sulfonatedaluminium phthalocyanines, xanthene dyes and mixtures thereof;

(2) preformed peracids: Suitable preformed peracids include, but are notlimited to, compounds selected from the group consisting ofpercarboxylic acids and salts, percarbonic acids and salts, perimidicacids and salts, peroxymonosulfuric acids and salts, for example,Oxone®, and mixtures thereof. Suitable percarboxylic acids includehydrophobic and hydrophilic peracids having the formula R—(C═O)O—O-Mwherein R is an alkyl group, optionally branched, having, when theperacid is hydrophobic, from 6 to 14 carbon atoms, or from 8 to 12carbon atoms and, when the peracid is hydrophilic, less than 6 carbonatoms or even less than 4 carbon atoms; and M is a counterion, forexample, sodium, potassium or hydrogen;

(3) sources of hydrogen peroxide, for example, inorganic perhydratesalts, including alkali metal salts such as sodium salts of perborate(usually mono- or tetra-hydrate), percarbonate, persulphate,perphosphate, persilicate salts and mixtures thereof. In one aspect ofthe invention the inorganic perhydrate salts are selected from the groupconsisting of sodium salts of perborate, percarbonate and mixturesthereof. When employed, inorganic perhydrate salts are typically presentin amounts of from 0.05 to 40 wt %, or 1 to 30 wt % of the overallfabric and home care product and are typically incorporated into suchfabric and home care products as a crystalline solid that may be coated.Suitable coatings include, inorganic salts such as alkali metalsilicate, carbonate or borate salts or mixtures thereof, or organicmaterials such as water-soluble or dispersible polymers, waxes, oils orfatty soaps; and

(4) bleach activators having R—(C═O)-L wherein R is an alkyl group,optionally branched, having, when the bleach activator is hydrophobic,from 6 to 14 carbon atoms, or from 8 to 12 carbon atoms and, when thebleach activator is hydrophilic, less than 6 carbon atoms or even lessthan 4 carbon atoms; and L is leaving group. Examples of suitableleaving groups are benzoic acid and derivatives thereof—especiallybenzene sulphonate. Suitable bleach activators include dodecanoyloxybenzene sulphonate, decanoyl oxybenzene sulphonate, decanoyloxybenzoic acid or salts thereof, 3,5,5-trimethyl hexanoyloxybenzenesulphonate, tetraacetyl ethylene diamine (TAED) and nonanoyloxybenzenesulphonate (NOBS). Suitable bleach activators are also disclosed in WO98/17767. While any suitable bleach activator may be employed, in oneaspect of the invention the subject fabric and home care product maycomprise NOBS, TAED or mixtures thereof.

When present, the peracid and/or bleach activator is generally presentin the fabric and home care product in an amount of from about 0.1 toabout 60 wt %, from about 0.5 to about 40 wt % or even from about 0.6 toabout 10 wt % based on the fabric and home care product. One or morehydrophobic peracids or precursors thereof may be used in combinationwith one or more hydrophilic peracid or precursor thereof.

The amounts of hydrogen peroxide source and peracid or bleach activatormay be selected such that the molar ratio of available oxygen (from theperoxide source) to peracid is from 1:1 to 35:1, or even 2:1 to 10:1.

Surfactants—The fabric and home care products according to the presentinvention may comprise a surfactant or surfactant system wherein thesurfactant can be selected from nonionic surfactants, anionicsurfactants, cationic surfactants, ampholytic surfactants, zwitterionicsurfactants, semi-polar nonionic surfactants and mixtures thereof. Whenpresent, surfactant is typically present at a level of from about 0.1%to about 60%, from about 1% to about 50% or even from about 5% to about40% by weight of the subject fabric and home care product.

Builders—The fabric and home care products of the present invention maycomprise one or more detergent builders or builder systems. When abuilder is used, the subject fabric and home care product will typicallycomprise at least about 1%, from about 5% to about 60% or even fromabout 10% to about 40% builder by weight of the subject fabric and homecare product.

Builders include, but are not limited to, the alkali metal, ammonium andalkanolammonium salts of polyphosphates, alkali metal silicates,alkaline earth and alkali metal carbonates, aluminosilicate builders andpolycarboxylate compounds, ether hydroxypolycarboxylates, copolymers ofmaleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulphonic acid, and carboxymethyloxysuccinic acid, thevarious alkali metal, ammonium and substituted ammonium salts ofpolyacetic acids such as ethylenediamine tetraacetic acid andnitrilotriacetic acid, as well as polycarboxylates such as melliticacid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid,benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, andsoluble salts thereof.

Chelating Agents—The fabric and home care products herein may contain achelating agent. Suitable chelating agents include copper, iron and/ormanganese chelating agents and mixtures thereof. When a chelating agentis used, the subject fabric and home care product may comprise fromabout 0.005% to about 15% or even from about 3.0% to about 10% chelatingagent by weight of the subject fabric and home care product. Suitablechelants include DTPA (Diethylene triamine pentaacetic acid), HEDP(Hydroxyethane diphosphonic acid), DTPMP (Diethylene triaminepenta(methylene phosphonic acid)), 1,2-Dihydroxybenzene-3,5-disulfonicacid disodium salt hydrate, ethylenediamine, diethylene triamine,ethylenediaminedisuccinic acid (EDDS),N-hydroxyethylethylenediaminetri-acetic acid (HEDTA),triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiaceticacid (HEIDA), dihydroxyethylglycine (DHEG),ethylenediaminetetrapropionic acid (EDTP) and derivatives thereof.

Dye Transfer Inhibiting Agents—The fabric and home care products of thepresent invention may also include one or more dye transfer inhibitingagents. Suitable polymeric dye transfer inhibiting agents include, butare not limited to, polyvinylpyrrolidone polymers, polyamine N-oxidepolymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole,polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. Whenpresent in a subject fabric and home care product, the dye transferinhibiting agents may be present at levels from about 0.0001% to about10%, from about 0.01% to about 5% or even from about 0.1% to about 3% byweight of the fabric and home care product.

Brighteners—The fabric and home care products of the present inventioncan also contain additional components that may tint articles beingcleaned, such as fluorescent brighteners. Suitable fluorescentbrightener levels include lower levels of from about 0.01, from about0.05, from about 0.1 or even from about 0.2 wt % to upper levels of 0.5or even 0.75 wt %.

Dispersants—The fabric and home care products of the present inventioncan also contain dispersants. Suitable water-soluble organic materialsinclude the homo- or co-polymeric acids or their salts, in which thepolycarboxylic acid comprises at least two carboxyl radicals separatedfrom each other by not more than two carbon atoms.

Enzyme Stabilizers—Enzymes for use in detergents can be stabilized byvarious techniques. The enzymes employed herein can be stabilized by thepresence of water-soluble sources of calcium and/or magnesium ions inthe finished fabric and home care products that provide such ions to theenzymes. In case of aqueous fabric and home care products comprisingprotease, a reversible protease inhibitor, such as a boron compound, orcompounds such as calcium formate, sodium formate and 1,2-propane diolcan be added to further improve stability.

Catalytic Metal Complexes—Applicants' compositions may include catalyticmetal complexes. One type of metal-containing bleach catalyst is acatalyst system comprising a transition metal cation of defined bleachcatalytic activity, such as copper, iron, titanium, ruthenium, tungsten,molybdenum, or manganese cations, an auxiliary metal cation havinglittle or no bleach catalytic activity, such as zinc or aluminumcations, and a sequestrate having defined stability constants for thecatalytic and auxiliary metal cations, particularlyethylenediaminetetraacetic acid,ethylenediaminetetra(methylenephosphonic acid) and water-soluble saltsthereof. Such catalysts are disclosed in U.S. Pat. No. 4,430,243.

-   If desired, the compositions herein can be catalyzed by means of a    manganese compound. Such compounds and levels of use are well known    in the art and include, for example, the manganese-based catalysts    disclosed in U.S. Pat. No. 5,576,282.-   Cobalt bleach catalysts useful herein are known, and are described,    for example, in U.S. Pat. Nos. 5,597,936; 5,595,967. Such cobalt    catalysts are readily prepared by known procedures, such as taught    for example in U.S. Pat. Nos. 5,597,936, and 5,595,967.-   Compositions herein may also suitably include a transition metal    complex of ligands such as bispidones (WO 05/042532 A1) and/or    macropolycyclic rigid ligands—abbreviated as “MRLs”. As a practical    matter, and not by way of limitation, the compositions and processes    herein can be adjusted to provide on the order of at least one part    per hundred million of the active MRL species in the aqueous washing    medium, and will typically provide from about 0.005 ppm to about 25    ppm, from about 0.05 ppm to about 10 ppm, or even from about 0.1 ppm    to about 5 ppm, of the MRL in the wash liquor.-   Suitable transition-metals in the instant transition-metal bleach    catalyst include, for example, manganese, iron and chromium.    Suitable MRLs include    5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane.-   Suitable transition metal MRLs are readily prepared by known    procedures, such as taught for example in WO 00/32601, and U.S. Pat.    No. 6,225,464.-   Solvents—Suitable solvents include water and other solvents such as    lipophilic fluids. Examples of suitable lipophilic fluids include    siloxanes, other silicones, hydrocarbons, glycol ethers, glycerine    derivatives such as glycerine ethers, perfluorinated amines,    perfluorinated and hydrofluoroether solvents, low-volatility    nonfluorinated organic solvents, diol solvents, other    environmentally-friendly solvents and mixtures thereof.    Processes of Making Fabric and Home Care Products

The fabric and home care products of the present invention can beformulated into any suitable form and prepared by any process chosen bythe formulator, non-limiting examples of which are described inApplicants' examples and in U.S. Pat. No. 4,990,280; U.S. 20030087791A1;U.S. 20030087790A1; U.S. 20050003983A1; U.S. 20040048764A1; U.S. Pat.Nos. 4,762,636; 6,291,412; U.S. 20050227891A1; EP 1070115A2; U.S. Pat.Nos. 5,879,584; 5,691,297; 5,574,005; 5,569,645; 5,565,422; 5,516,448;5,489,392; 5,486,303 all of which are incorporated herein by reference.

Method of Use

The present invention includes a method for cleaning and/or treating asitus inter alia a surface or fabric. In one aspect, such methodcomprises the steps of optionally washing and/or rinsing said surface orfabric, contacting said surface or fabric with any fabric and home careproduct disclosed in this specification then optionally washing and/orrinsing said surface or fabric is disclosed.

As used herein, washing includes but is not limited to, scrubbing, andmechanical agitation. Drying of such surfaces or fabrics may beaccomplished by any one of the common means employed either in domesticor industrial settings. Such means include but are not limited to forcedair or still air drying at ambient or elevated temperatures at pressuresbetween 5 and 0.01 atmospheres in the presence or absence ofelectromagnetic radiation, including sunlight, infrared, ultraviolet andmicrowave irradiation. In one aspect, said drying may be accomplished attemperatures above ambient by employing an iron wherein, for example,said fabric may be in direct contact with said iron for relatively shortor even extended periods of time and wherein pressure may be exertedbeyond that otherwise normally present due to gravitational force. Inanother aspect, said drying may be accomplished at temperatures aboveambient by employing a dryer. Apparatus for drying fabric is well knownand it is frequently referred to as a clothes dryer. In addition toclothes such appliances are used to dry many other items includingtowels, sheets, pillowcases, diapers and so forth and such equipment hasbeen accepted as a standard convenience in many nations of the worldsubstantially replacing the use of clothes lines for drying of fabric.Most dryers in use today use heated air which is passed over and orthrough the fabric as it is tumbled within the dryer. The air may beheated, for example, either electronically, via gas flame, or even withmicrowave radiation. Such air may be heated from about 15° C. to about400° C., from about 25° C. to about 200° C., from about 35° C. to about100° C., or even from about 40° C. to about 85° C. and used in the dryerto dry a surface and/or a fabric. As will be appreciated by one skilledin the art, the cleaning compositions of the present invention areideally suited for use in laundry applications. Accordingly, the presentinvention includes a method for laundering a fabric. The methodcomprises the steps of contacting a fabric to be laundered with a saidcleaning laundry solution comprising at least one embodiment ofApplicants' cleaning composition, cleaning additive or mixture thereof.The fabric may comprise most any fabric capable of being laundered innormal consumer or institutional use conditions. The solution preferablyhas a pH of from about 8 to about 10.5. The compositions may be employedat concentrations of from about 500 ppm to about 15,000 ppm in solution.The water temperatures typically range from about 5° C. to about 90° C.The water to fabric ratio is typically from about 1:1 to about 30:1.

Test Methods

-   Test Method 1

A protocol to define whether a dye or pigment material is a fabrichueing agent for the purpose of the invention is given here:

-   1.) Fill two tergotometer pots with 800 ml of Newcastle upon Tyne,    UK, City Water (˜12 grains per US gallon total hardness, supplied by    Northumbrian Water, Pity Me, Durham, Co. Durham, UK).-   2) Insert pots into tergotometer, with water temperature controlled    at 30° C. and agitation set at 40 rpm for the duration of the    experiment.-   3) Add 4.8 g of IEC-B detergent (IEC 60456 Washing Machine Reference    Base Detergent Type B), supplied by wfk, Brüggen-Bracht, Germany, to    each pot.-   4) After two minutes, add 2.0 mg active colorant to the first pot.-   5) After one minute, add 50 g of flat cotton vest (supplied by    Warwick Equest, Consett, County Durham, UK), cut into 5 cm×5 cm    swatches, to each pot.-   6) After 10 minutes, drain the pots and re-fill with cold Water (16°    C.) having a water hardness of 14.4 English Clark Degrees Hardness    with a 3:1 Calcium to Magnesium molar ratio.-   7) After 2 minutes rinsing, remove fabrics.-   8) Repeat steps 3-7 for a further three cycles using the same    treatments.-   9) Collect and line dry the fabrics indoors for 12 hours.-   10) Analyse the swatches using a Hunter Miniscan spectrometer fitted    with D65 illuminant and UVA cutting filter, to obtain Hunter a    (red-green axis) and Hunter b (yellow-blue axis) values.-   11) Average the Hunter a and Hunter b values for each set of    fabrics. If the fabrics treated with colorant under assessment show    an average difference in hue of greater than 0.2 units on either the    a axis or b axis, it is deemed to be a fabric hueing agent for the    purpose of the invention.-   Test Method 2—For Test Method 2, the BMI microswatch assay is run    using the detergent compositions 1, 2 or 4 from Table 1-3. The    detergent is dissolved in water that has a hardness of 6 gpg and    adjusted to a temperature of 16° C. Performance of the variant    enzymes is then determined as per the BMI microswatch assay    described. The performance index is determined by comparing the    performance of the variant with that of the enzyme of SEQ ID NO:2,    SEQ ID NO:4 or SEQ ID NO:6, with in all cases the enzyme dosage    being 1 ppm. This Method is described in further detail in Example    31.-   Test Method 3—For Test Method 3 the BMI microswatch assay is run    using the detergent compositions 1, 2 or 4 from Table 1-3. The    detergent is dissolved in water that has a hardness of 6 gpg and    adjusted to a temperature of 16° C. Performance of the variant    enzymes is then determined as per the BMI microswatch assay    described. The performance index is determined by comparing the    performance of the variant with that of the enzyme of SEQ ID NO:2,    SEQ ID NO:4 or SEQ ID NO:6, with in all cases the enzyme dosage    being 0.4 ppm. This Method is described in further detail in Example    1.-   Test Method 4—For Test Method 4, the BMI microswatch assay is run    using the detergent composition 10 from Table 19-4. The detergent is    dissolved in water that has a hardness of 12 gpg and adjusted to a    temperature of 16° C. Performance of the variant enzymes is then    determined as per the BMI microswatch assay described. The    performance index is determined by comparing the performance of the    variant with that of the enzyme of SEQ ID NO:755, with in all cases    the enzyme dosage being 1.6 ppm. Enzymes having a performance index    of 1.1 or greater are viewed to be Series 1 GG36 cold water    proteases. This Method is described in further detail in Example 19.-   Test Method 5—For Test Method 5, the BMI microswatch assay is run    using the detergent composition 7 from Table 19-4. The detergent is    dissolved in water that has a hardness of 6 gpg and adjusted to a    temperature of 16° C. Performance of the variant enzymes is then    determined as per the BMI microswatch assay described. The    performance index is determined by comparing the performance of the    variant with that of the enzyme of SEQ ID NO:755, with in all cases    the enzyme dosage being 4 ppm. Enzymes having a performance index of    1.1 or greater are viewed to be Series 1 GG36 cold water proteases.    This Method is described in further detail in Example 19.-   Test Method 6—For Test Method 6 the BMI microswatch assay is run    using the detergent composition 7 from Table 19-4. The detergent is    dissolved in water that has a hardness of 6 gpg and adjusted to a    temperature of 16° C. Performance of the variant enzymes is then    determined as per the BMI microswatch assay described. The    performance index is determined by comparing the performance of the    variant with that of a reference enzyme, said reference enzyme being    the enzyme of SEQ ID NO:755 consisting the A158E mutation, with in    all cases the enzyme dosage being 4 ppm. Enzymes having a    performance index of 1.0 or greater are viewed to be Series 1

GG36 cold water proteases. This Method is described in further detail inExample 19.

-   Test Method 7—Electrical conductivity of an aqueous solution is    assayed according to the standard method ASTM D1125 and reported in    units of milliSiemens/cm, abbreviated to mS/cm in this patent.

Experimental

In the experimental disclosure which follow, the following abbreviationsapply: PI (Performance Index), ppm (parts per million); M (molar); mM(millimolar); μM (micromolar); nM (nanomolar); mol (moles); mmol(millimoles); μmol (micromoles); nmol (nanomoles); gm (grams); mg(milligrams); μg (micrograms); pg (picograms); L (liters); ml and mL(milliliters); μl and μL (microliters); cm (centimeters); mm(millimeters); μm (micrometers); nm (nanometers); U (units); V (volts);MW (molecular weight); sec (seconds); min(s) (minute/minutes); h(s) andhr(s) (hour/hours); ° C. (degrees Centigrade); QS (quantity sufficient);ND (not done); rpm (revolutions per minute); GH (degrees Germanhardness); H₂O (water); dH₂O (deionized water); HCl (hydrochloric acid);aa (amino acid); bp (base pair); kb (kilobase pair); kD (kilodaltons);cDNA (copy or complementary DNA); DNA (deoxyribonucleic acid); ssDNA(single stranded DNA); dsDNA (double stranded DNA); dNTP(deoxyribonucleotide triphosphate); RNA (ribonucleic acid); MgCl₂(magnesium chloride); NaCl (sodium chloride); w/v (weight to volume);v/v (volume to volume); w/w (weight to weight); g (gravity); OD (opticaldensity); ppm (parts per million); Dulbecco's phosphate bufferedsolution (DPBS); SOC (2% Bacto-Tryptone, 0.5% Bacto Yeast Extract, 10 mMNaCl, 2.5 mM KCl); Terrific Broth (TB; 12 g/l Bacto-Tryptone, 24 g/lglycerol, 2.31 g/l KH₂PO₄, and 12.54 g/l K₂HPO₄); OD₂₈₀ (optical densityat 280 nm); OD₆₀₀ (optical density at 600 nm); A₄₀₅ (absorbance at 405nm); Vmax (the maximum initial velocity of an enzyme catalyzedreaction); PAGE (polyacrylamide gel electrophoresis); PBS (phosphatebuffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]);PBST (PBS+0.25% TWEEN®-20); PEG (polyethylene glycol); PCR (polymerasechain reaction); RT-PCR (reverse transcription PCR); SDS (sodium dodecylsulfate); Tris (tris(hydroxymethyl)aminomethane); HEPES(N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS (HEPESbuffered saline); Tris-HCl(tris[Hydroxymethyl]aminomethane-hydrochloride); Tricine(N-[tris-(hydroxymethyl)-methyl]-glycine); CHES (2-(N-cyclo-hexylamino)ethane-sulfonic acid); TAPS(3-{[tris-(hydroxymethyl)-methyl]-amino}-propanesulfonic acid); CAPS(3-(cyclo-hexylamino)-propane-sulfonic acid; DMSO (dimethyl sulfoxide);DTT (1,4-dithio-DL-threitol); SA (sinapinic acid(s,5-dimethoxy-4-hydroxy cinnamic acid); TCA (trichloroacetic acid);Glut and GSH (reduced glutathione); GSSG (oxidized glutathione); TCEP(Tris[2-carboxyethyl] phosphine); Ci (Curies); mCi (milliCuries); μCi(microCuries); HPLC (high-performance liquid chromatography); RP-HPLC(reverse phase high pressure liquid chromatography); TLC (thin layerchromatography); MALDI-TOF (matrix-assisted laserdesorption/ionization—time of flight); Ts (tosyl); Bn (benzyl); Ph(phenyl); Ms (mesyl); Et (ethyl), Me (methyl); Taq (Thermus aquaticusDNA polymerase); Klenow (DNA polymerase I large (Klenow) fragment); EGTA(ethylene glycol-bis(ß-aminoethyl ether) N,N,N′,N′-tetraacetic acid);EDTA (ethylenediaminetetracetic acid); bla (β-lactamase orampicillin-resistance gene); HDL (heavy duty liquid); HDD (heavy dutypowder detergent); HSG (high suds granular detergent); CEE (Central andEastern Europe); WE (Western Europe); NA, when used in reference todetergents (North America); Japan and JPN, when used in reference todetergents (Japan); MTP (microtiter plate); MJ Research (MJ Research,Reno, Nev.); Baseclear (Baseclear BV, Inc., Leiden, The Netherlands);PerSeptive (PerSeptive Biosystems, Framingham, Mass.); ThermoFinnigan(ThermoFinnigan, San Jose, Calif.); Argo (Argo BioAnalytica, MorrisPlains, N.J.); Seitz EKS (SeitzSchenk Filtersystems GmbH, Bad Kreuznach,Germany); Pall (Pall Corp., East Hills, N.Y. and Bad Kreuznach,Germany); Spectrum (Spectrum Laboratories, Dominguez Rancho, Calif.);Molecular Structure (Molecular Structure Corp., Woodlands, Tex.);Accelrys (Accelrys, Inc., San Diego, Calif.); Chemical Computing(Chemical Computing Corp., Montreal, Canada); New Brunswick (NewBrunswick Scientific, Co., Edison, N.J.); CFT (Center for TestMaterials, Vlaardingen, The Netherlands); P&G and Proctor & Gamble(Proctor & Gamble, Inc., Cincinnati, Ohio); GE Healthcare (GEHealthcare, Chalfont St. Giles, United Kingdom); DNA2.0 (DNA2.0, MenloPark, Calif.); OXOID (Oxoid, Basingstoke, Hampshire, UK); Megazyme(Megazyme International Ireland Ltd., Bray Business Park, Bray, Co.,Wicklow, Ireland); Finnzymes (Finnzymes Oy, Espoo, Finland); Kelco (CPKelco, Wilmington, Del.); Corning (Corning Life Sciences, Corning,N.Y.); (NEN (NEN Life Science Products, Boston, Mass.); Pharma AS(Pharma AS, Oslo, Norway); Dynal (Dynal, Oslo, Norway); Bio-Synthesis(Bio-Synthesis, Lewisville, Tex.); ATCC (American Type CultureCollection, Rockville, Md.); Gibco/BRL (Gibco/BRL, Grand Island, N.Y.);Sigma (Sigma Chemical Co., St. Louis, Mo.); Pharmacia (PharmaciaBiotech, Piscataway, N.J.); NCBI (National Center for BiotechnologyInformation); Applied Biosystems (Applied Biosystems, Foster City,Calif.); BD Biosciences and/or Clontech (BD Biosciences CLONTECHLaboratories, Palo Alto, Calif.); Operon Technologies (OperonTechnologies, Inc., Alameda, Calif.); MWG Biotech (MWG Biotech, HighPoint, N.C.); Oligos Etc (Oligos Etc. Inc, Wilsonville, Oreg.); Bachem(Bachem Bioscience, Inc., King of Prussia, Pa.); Difco (DifcoLaboratories, Detroit, Mich.); Mediatech (Mediatech, Herndon, Va.; SantaCruz (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.); Oxoid (OxoidInc., Ogdensburg, N.Y.); Worthington (Worthington Biochemical Corp.,Freehold, N.J.); GIBCO BRL or Gibco BRL (Life Technologies, Inc.,Gaithersburg, Md.); Millipore (Millipore, Billerica, Mass.); Bio-Rad(Bio-Rad, Hercules, Calif.); Invitrogen (Invitrogen Corp., San Diego,Calif.); NEB (New England Biolabs, Beverly, Mass.); Sigma (SigmaChemical Co., St. Louis, Mo.); Pierce (Pierce Biotechnology, Rockford,Ill.); Takara (Takara Bio Inc. Otsu, Japan); Roche (Hoffmann-La Roche,Basel, Switzerland); Gene Oracle (Gene Oracle, Inc., Mountain View,Calif.); EM Science (EM Science, Gibbstown, N.J.); Qiagen (Qiagen, Inc.,Valencia, Calif.); Biodesign (Biodesign Intl., Saco, Me.); Aptagen(Aptagen, Inc., Herndon, Va.); Sorvall (Sorvall brand, from KendroLaboratory Products, Asheville, N.C.); Molecular Devices (MolecularDevices, Corp., Sunnyvale, Calif.); R&D Systems (R&D Systems,Minneapolis, Minn.); Siegfried Handel (Siegfried Handel AG, Zofingen,Switzerland); Stratagene (Stratagene Cloning Systems, La Jolla, Calif.);Marsh (Marsh Biosciences, Rochester, N.Y.); Geneart (Geneart GmbH,Regensburg, Germany); Bio-Tek (Bio-Tek Instruments, Winooski, Vt.);(Biacore (Biacore, Inc., Piscataway, N.J.); PeproTech (PeproTech, RockyHill, N.J.); SynPep (SynPep, Dublin, Calif.); New Objective (NewObjective brand; Scientific Instrument Services, Inc., Ringoes, N.J.);Waters (Waters, Inc., Milford, Mass.); Matrix Science (Matrix Science,Boston, Mass.); Dionex (Dionex, Corp., Sunnyvale, Calif.); Monsanto(Monsanto Co., St. Louis, Mo.); Wintershall (Wintershall AG, Kassel,Germany); BASF (BASF Co., Florham Park, N.J.); Huntsman (HuntsmanPetrochemical Corp., Salt Lake City, Utah); Shell Chemicals (ShellChemicals, Inc., London, UK); Stepan (Stepan, Northfield, Ill.);Clariant (Clariant, Sulzbach, Germany); Industrial Zeolite (IndustrialZeolite Ltd., Grays, Essex, UK); Jungbunzlauer (Jungbunzlauer, Basel,Switzerland); Solvay (Solvay, Brussels, Belgium); 3V Sigma (3V Sigma,Bergamo, Italy); Innospec (Innospec, Ellesmere Port, UK); Thermphos(Thermphos, Vlissiggen-Ost, The Netherlands); Ciba Specialty (CibaSpecialty Chemicals, Basel, Switzerland); Dow Corning (Dow Corning,Barry, UK); Enichem (Enichem Iberica, Barcelona, Spain); Fluka Chemie AG(Fluka Chemie AG, Buchs, Switzerland); Gist-Brocades (Gist-Brocades, NV,Delft, The Netherlands); Dow Corning (Dow Corning Corp., Midland,Mich.); Mettler-Toledo (Mettler-Toledo Inc, Columbus, Ohio); RB(Reckitt-Benckiser, Slough, UK); and Microsoft (Microsoft, Inc.,Redmond, Wash.).

For North American (NA) and Western European (WE) heavy duty liquidlaundry (HDL) detergents, heat inactivation of the enzymes present incommercially-available detergents is performed by placing pre-weighedliquid detergent (in a glass bottle) in a water bath at 95° C. for 2hours. The incubation time for heat inactivation of NA and WE auto dishwashing (ADW) detergents is 8 hours. Both un-heated and heateddetergents are assayed within 5 minutes of dissolving the detergent toaccurately determine percentage deactivated. Enzyme activity is testedby the AAPF assay.

For testing of enzyme activity in heat-inactivated detergents, workingsolutions of detergents are made from the heat inactivated stocks.Appropriate amounts of water hardness (e.g., 6 gpg or 12 gpg) and bufferare added to the detergent solutions to match the desired conditions.The solutions are mixed by vortexing or inverting the bottles. Thefollowing Table provides information regarding some of thecommercially-available detergents and test conditions used herein. Insome experiments, additional and/or other commercially availabledetergents find use in the following Examples.

TABLE 1.1 Laundry and Dish Washing Conditions Region Form DoseDetergent* Buffer gpg pH T (° C.) Laundry (Heavy Duty Liquid andGranular) NA HDL 0.78 g/l   P&G TIDE ® 2X 5 mM HEPES 6 8.0 20 WE HDL 5.0g/L Henkel PERSIL ™ 5 mM HEPES 12 8.2 40 WE HDG 8.0 g/L P&G ARIEL ® 2 mMNa₂ CO₃ 12 10.5 40 JPN HDG 0.7 g/L P&G TIDE ® 2 mM Na₂ CO₃ 6 10.0 20 NAHDG 1.0 g/L P&G TIDE ® 2 mM Na₂ CO₃ 6 10.0 20 Automatic Dish Washing WEADW 3.0 g/L RB CALGONIT ™ 2 mM Na₂ CO₃ 21 10.0 40 NA ADW 3.0 g/L P&GCASCADE ® 2 mM Na₂ CO₃ 9 10.0 40

In some additional aspects, the following solutions find use:

TABLE 1-2 Working Detergent Solutions Temp Detergent Detergent (C.) g/LpH Buffer gpg TIDE ® 2X Cold 16 0.98 8 5 mM HEPES 6 TIDE ® 2X Cold 320.98 8 5 mM HEPES 6 TIDE ® 2X Cold 16 0.98 7 5 mM MOPS 6

The examples, which are intended to be purely exemplary of the inventionand should therefore not be considered to limit the invention in anyway, also describe and detail aspects and embodiments of the inventiondiscussed above. The foregoing examples and detailed description areoffered by way of illustration and not by way of limitation.

BPN′ Variant or Part I Examples

Table of Detergents

The compositions of the detergents used in the assays in BPN′ Variant(or Part I) Examples are shown in Table 1-3. BPN′ variant proteinsamples were added to the detergent compositions as described in Part IExample 1 to assay for the various properties tested.

The following are liquid laundry detergent compositions suitable fortop-loading automatic washing machines (1, 2 & 4) and front loadingwashing machines (3).

TABLE 1-3 Composition of Detergents Used in the Assays to Test BPN′Variants Composition (wt % of composition) Ingredient 1 2 3 4 C₁₂₋₁₅Alkylethoxy(1.8)sulfate 14.7 11.6 16.31 C_(11.8) Alkylbenzene sulfonate4.3 11.6 8.3 7.73 C₁₆₋₁₇ Branched alkyl sulfate 1.7 1.29 3.09 C₁₂₋₁₄Alkyl-9-ethoxylate 0.9 1.07 1.31 C₁₂ dimethylamine oxide 0.6 0.64 1.03Citric acid 3.5 0.65 3 0.66 C₁₂₋₁₈ fatty acid 1.5 2.32 3.6 1.52 SodiumBorate (Borax) 2.5 2.46 1.2 2.53 Sodium C₁₂₋₁₄ alkyl ethoxy 3 sulfate2.9 C₁₄₋₁₅ alkyl 7-ethoxylate 4.2 C₁₂₋₁₄ Alkyl-7-ethoxylate 1.7 Caformate 0.09 0.09 0.09 A compound having the following generalstructure: 1.2 bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)-bis((C₂H₅O)(C₂H₄O)n), wherein n = from 20 to 30, and x = from 3 to 8, orsulphated or sulphonated variants thereof Random graft co-polymer¹ 1.460.5 Ethoxylated Polyethylenimine ² 1.5 1.29 1.44 Diethylene triaminepentaacetic acid 0.34 0.64 0.34 Diethylene triamine penta(methylenephosphonic acid) 0.3 Tinopal AMS-GX 0.06 Tinopal CBS-X 0.2 0.17 0.29Amphiphilic alkoxylated grease cleaning polymer ³ 1.28 1 0.4 1.93Ethanol 2 1.58 1.6 5.4 Propylene Glycol 3.9 3.59 1.3 4.3 Diethyleneglycol 1.05 1.54 1.15 Polyethylene glycol 0.06 0.04 0.1 Monoethanolamine3.05 2.41 0.4 1.26 NaOH 2.44 1.8 3.01 Sodium Cumene Sulphonate 1 SodiumFormate 0.11 0.09 Water, Aesthetics (Dyes, perfumes) and Minors balancebalance balance (Enzymes, solvents, structurants) ¹Random graftcopolymer is a polyvinyl acetate grafted polyethylene oxide copolymerhaving a polyethylene oxide backbone and multiple polyvinyl acetate sidechains. The molecular weight of the polyethylene oxide backbone is about6000 and the weight ratio of the polyethylene oxide to polyvinyl acetateis about 40 to 60 and no more than 1 grafting point per 50 ethyleneoxide units. ² Polyethylenimine (MW = 600) with 20 ethoxylate groups per—NH. ³ Amphiphilic alkoxylated grease cleaning polymer is apolyethylenimine (MW = 600) with 24 ethoxylate groups per —NH and 16propoxylate groups per —NHVectors, Cells, and Methods for Making Protease Variant Polypeptides ofthe Invention

A variety of methods are known in the art that are suitable forgenerating modified polynucleotides of the invention that encodeprotease variants of the invention (such as cold water proteases of theinvention), including, but not limited to, e.g., site-saturationmutagenesis, scanning mutagenesis, insertional mutagenesis, deletionmutagenesis, random mutagenesis, site-directed mutagenesis, anddirected-evolution, as well as various other recombinatorial approaches.Methods for making modified polynucleotides and proteins (e.g., proteasevariants) include DNA shuffling methodologies (see, e.g., Stemmer W P,Proc. Natl. Acad. Sci. USA 91(22):10747-51 (1994)); methods based onnon-homologous recombination of genes, e.g., ITCHY (Ostermeier et al.,Bioorg. Med. Chem. 7:2139-44 [1999]); SCRATCHY (Lutz et al. Proc. Natl.Acad. Sci. USA 98:11248-53 [2001]); SHIPREC (Sieber et al., Nat.Biotechnol. 19:456-60 [2001]); NRR (Bittker et al., Nat. Biotechnol.20:1024-9 [2001]; Bittker et al., Proc Natl. Acad. Sci. USA101:7011-6[2004]); methods that rely on the use of oligonucleotides to insertrandom and targeted mutations, deletions and/or insertions (Ness et al.,Nat. Biotechnol. 20:1251-5 [2002]; Coco et al., Nat. Biotechnol.20:1246-50 [2002]; Zha et al., Chembiochem. 4:34-9 [2003]; Glaser etal., J. Immunol. 149:3903-13 [1992]); see also Arkin and Youvan,Biotechnology 10:297-300 (1992); Reidhaar-Olson et al., Methods Enzymol.208:564-86 (1991).

In one aspect, a full-length parent polynucleotide is ligated into anappropriate expression plasmid, and the following mutagenesis method isused to facilitate the construction of the modified protease of thepresent invention, although other methods may be used. The method isbased on that described by Pisarchik et al. (Pisarchik et al., Prot.Eng. Des. Select. 20:257-265 [2007]). In one aspect, an added advantageis provided in that the restriction enzyme cuts outside its recognitionsequence, which allows digestion of practically any nucleotide sequenceand precludes formation of a restriction site scar.

In one approach, a naturally-occurring gene encoding a full-lengthprotease is obtained and sequenced and scanned for one or more points atwhich it is desired to make a mutation (e.g., deletion, insertion,substitution) at one or more amino acids. Mutation of the gene in orderto change its sequence to conform to the desired sequence isaccomplished by primer extension in accord with generally known methods.Fragments to the left and to the right of the desired point(s) ofmutation are amplified by PCR and to include the Eam1104I restrictionsite. The left and right fragments are digested with Eam1104I togenerate a plurality of fragments having complementary three baseoverhangs, which are then pooled and ligated to generate a library ofmodified sequences containing one or more mutations. This method avoidsthe occurrence of frame-shift mutations. This method also simplifies themutagenesis process because all of the oligonucleotides can besynthesized so as to have the same restriction site, and no syntheticlinkers are necessary to create the restriction sites as is required bysome other methods.

Part I Examples Example 1 Assays

Various assays were used as set forth below. Any deviations from theprotocols provided below are indicated in the subsequent Examples.

A. TCA Assay for Protein Content Determination in 96-Well MicrotiterPlates

For BPN′ and BPN′ variants, this assay was started using filtered B.subtilis bacterial culture supernatant from microtiter plates grown 3-4days at 33-37° C. with shaking at 230-250 rpm and humidified aeration. Afresh 96-well flat bottom microtiter plate (MTP) was used for the assay.First, 100 μL/well of 0.25 N HCl was placed in each well. Then, 25 μL offiltered culture broth was added. The light scattering/absorbance at 405nm (use 5 sec mixing mode in the plate reader) was then determined inorder to provide the “blank” reading. For the test, 100 μL/well of 30%(w/v) trichloroacetic acid (TCA) was placed in the plates and incubatedfor 10 minutes at room temperature. The light scattering/absorbance at405 nm (use 5 sec mixing mode in the plate reader) was then determined.The equipment used was a Biomek FX Robot (Beckman Coulter) and aSpectraMAX (type 340; Molecular Devices) MTP Reader; the MTPs were fromCostar (type 9017).

The calculations were performed by subtracting the blank (no TCA) fromthe test reading with TCA to provide a relative measure of the proteincontent in the samples. If desired, a standard curve can be created bycalibrating the TCA readings with AAPF assays of clones with knownconversion factors. However, the TCA results are linear with respect toprotein concentration from 250 to 2500 micrograms protein per ml (ppm)and can thus be plotted directly against enzyme performance for thepurpose of choosing good-performing variants. The turbidity/lightscatter increase in the samples correlates to the total amount ofprecipitable protein in the culture supernatant.

B. AAPF Protease Assay in 96-Well Microtiter Plates

In order to determine the protease activity of the proteases andvariants thereof of the present invention, the hydrolysis ofN-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-p-nitroanilide(suc-AAPF-pNA) was measured. The reagent solutions used were: 100 mMTris/HCl, pH 8.6, containing 0.005% TWEEN®-80 (Tris dilution buffer);100 mM Tris buffer, pH 8.6, containing 10 mM CaCl₂ and 0.005% TWEEN®-80(Tris/Ca buffer); and 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stocksolution) (Sigma: S-7388). To prepare a suc-AAPF-pNA working solution, 1ml suc-AAPF-pNA stock solution was added to 100 ml Tris/Ca buffer andmixed well for at least 10 seconds. The assay was performed by adding 10μl of diluted protease solution to each well, immediately followed bythe addition of 190 μl 1 mg/ml suc-AAPF-pNA working solution. Thesolutions were mixed for 5 sec., and the absorbance change in kineticmode (25 readings in 5 minutes) was read at 405 nm in an MTP reader, at25° C. The protease activity was expressed as AU (activity=ΔOD·min⁻¹ml⁻¹).

C. BMI Microswatch Assay of Test Method 3

Blood milk and ink (BMI) stained microswatches of 5.5 millimetercircular diameter were obtained from CFT. Before cutting the swatches,the fabric (EMPA 116) was washed with water. One microswatch was placedin each well of a 96-well non-binding microtiter plate (Corning 3641).The detergents used for the assays included Detergent Composition 1,Detergent Composition 2, and Detergent Composition 4. The detergentswere diluted in Milli-Q (deionized) water to a working strengthconcentration of 0.788 g/L. These detergents were buffered with 5 mMHEPES pH 8.2 or pH 7.2, which upon addition to detergent, buffers at pH8 or pH 7, respectively. Additionally, 6 grains per gallon (gpg) waterhardness (3:1 Ca:Mg—CaCl₂:MgCl₂.6H₂O) was added. The detergent solutionwas pre-equilibrated in an ice-water bath for 16° C. assays (roomtemperature for 32° C. assays) and pumped into a circulating reservoir(Beckman FX). Then, 190 μl of the desired detergent solution was addedto each well of the MTP that contained microswatches. To this mixture,10 μl of the diluted enzyme master dilution solution was added,providing an approximate enzyme concentration of 0.4-0.5 μg/mL. Themaster dilution was prepared from the culture supernatants at 8 μg/mL,where the approximate enzyme concentration of the culture supernatantsand BPN′-v3 or BPN′-v36 parent controls was determined using the AAPFprotease activity assay, basing the concentration on a purified BPN′-v3or BPN′-v36 standard of known concentration. The MTP was sealed withtape and placed in the iEMS incubator/shaker (Thermo/Labsystems) pre-setat 16° C. in a refrigerated dairy case or at 32° C. on the benchtop for20 minutes, with agitation at 1400 rpm. Following incubation under theappropriate conditions, the sealing tape was removed from each plate and125 μl (150 μl if pipetting by hand for smaller screens) of the solutionfrom each well was transferred into a fresh MTP (Corning 9017). The newMTP containing 125 μl-150 μl of solution/well was read at 600 nm (with 5sec mixing mode in the plate reader) using a MTP SpectraMax reader (type340; Molecular Devices). Blank controls containing a microswatch anddetergent but no enzyme were also included. The absorbance valueobtained was corrected for the blank value (substrate without enzyme),providing a measure of hydrolytic activity. For each sample (variant),the performance index was calculated as described below. This BMIMicroswatch Assay, run at 60° F. (16° C.) and pH 8, is referred toherein as “Test Method. 3”

D. Stability Assay

The stability of protease variants was determined in the presence of 40%concentrated Detergent Composition 3 diluted in water. The reagents usedwere Detergent Composition 3 diluted to 50% in Milli-Q water, 10 mM MES0.01% TWEEN®-80 pH 5.8 master dilution buffer, AAPF reagents: seeprotocol AAPF assay. The equipment used was F-bottom MTP (Corning 9017)for dilution of diluted enzyme into detergent as well as forsuc-AAPF-pNA plates, Biomek FX (Beckman Coulter), Spectramax Plus 384MTP Reader (Molecular Devices), iEMS Incubator/Shaker (1 mm amplitude)(Thermo Electron Corporation), sealing tape: Nunc (236366), circulatingreservoir (Beckman Fx).

Detergent Composition 3 was initially diluted to 50% in water. Thisdetergent was kept at room temperature and cycled through thecirculating reservoir. The iEMS incubators/shakers (Thermo/Labsystems)were pre-set at 43° C. Culture supernatants were diluted into platescontaining master dilution buffer to a concentration of ˜20 ppm (masterdilution plate). Then, 40 μl of sample from the master dilution platewas added to plates containing 160 μl 50% Detergent Composition 3 togive a final incubation concentration of 4 ppm. The contents were mixedand kept at room temperature and triplicate AAPF assays were performedimmediately on these plates and recorded as unstressed reads. The AAPFassay was modified such that 20 μL of sample from the step above wasadded to 190 μL of suc-AAPF-pNA working solution. The plates wereimmediately covered with sealing tape and placed in 43° C. iEMS shakersfor 30 min at 650 rpm. Following 30 minutes of incubation, triplicateAAPF assays were performed on these stress plates and recorded asstressed reads. The stability of the samples was determined bycalculating the ratio of the residual and initial AAPF activity asfollows: Residual Activity (%)=[mOD·min⁻¹ stressed]*100/[mOD·min⁻¹unstressed]. For each sample (variant), the performance index wascalculated as described below.

E. LAS/EDTA Stability Assay

The stability of protease variants in the presence of a representativeanionic surfactant (LAS=linear alkylbenzene sulfonate, specifically,sodium dodecylbenzenesulfonate-DOBS) and di-sodium EDTA was measuredafter incubation under defined conditions and the residual activity wasdetermined using the AAPF assay. The reagents used were dodecyllbenzenesulfonate, sodium salt (DOBS, Sigma No. D-2525), TWEEN®-80 (Sigma No.P-8074), di-sodium EDTA (Siegfried Handel No. 164599-02), HEPES (SigmaNo. H-7523), unstressed buffer: 50 mM HEPES (11.9 g/l)+0.005% TWEEN®-80,pH 8.0, Stress buffer: 50 mM HEPES (11.9 g/l), 0.1% (w/v) DOBS (1 g/l),10 mM EDTA (3.36 g/l), pH 8.0, reference protease and protease variantculture supernatants, containing 200-400 μg/ml protein. The equipmentused was V- or U-bottom MTPs as dilution plates (Greiner 651101 and650161, respectively), F-bottom MTPs (Corning 9017) for unstressed andLAS/EDTA buffer as well as for suc-AAPF-pNA plates, Biomek FX (BeckmanCoulter), Spectramax Plus 384 MTP Reader (Molecular Devices), iEMSIncubator/Shaker (1 mm amplitude) (Thermo Electron Corporation), andNunc sealing tape (236366).

The iEMS incubator/shaker (Thermo/Labsystems) was set at 29° C. Culturesupernatants were diluted into plates containing unstressed buffer to aconcentration of ˜25 ppm (master dilution plate). Then, 20 μl of samplefrom the master dilution plate was added to plates containing 180 μlunstressed buffer to give a final incubation concentration of 2.5 ppm.The contents were mixed and kept at room temperature and an AAPF assaywas performed on this plate. Then, 20 μl of sample from the masterdilution plate was also added to plates containing 180 μl stress buffer(50 mM HEPES (11.9 g/l), 0.1% (w/v) DOBS (1 g/l), 10 mM EDTA (3.36 g/l),pH 8.0). The solutions were mixed and immediately placed in 29° C. iEMSshaker for 30 min at 400 rpm. Following 30 minutes of incubation, anAAPF assay was performed on the stress plate. The stability of thesamples was determined by calculating the ratio of the residual andinitial AAPF activity as follows: Residual Activity (%)=[mOD·min−1stressed]*100/[mOD·min−1 unstressed]. For each sample (variant), theperformance index was calculated as described below.

Performance Index

The performance index provides a comparison of the performance of avariant (actual value) and a standard or reference protease enzyme(theoretical value) at the same protein concentration. The theoreticalvalues can be calculated using the parameters of a performance doseresponse curve (i.e. using a Langmuir equation to generate theperformance curve) of the standard/reference protease. A performanceindex (PI) that is greater than 1 (PI>1) identifies a better variant ascompared to the standard or reference protease (which may be, e.g.,wild-type protease or another protease variant), while a PI of 1 (PI=1)identifies a variant that performs the same as the standard or referenceprotease, and a PI that is less than 1 (PI<1) identifies a variant thatperforms worse than the standard or reference protease. Thus, the PIidentifies winners (e.g., variants having enhanced proteolytic activitycompared to that of the standard/reference protease) as well as variantsthat may be less desirable for use under certain circumstances (e.g.,variants having proteolytic activity lower than the proteolytic activityof the standard/reference protease).

It is important to note that protease variants of the invention havingperformance index values lower than that of a reference or standardprotease are nevertheless useful in the applications and methodsdescribed herein. For example, protease variants of the invention havingperformance index values lower than that of a reference or standardprotease have proteolytic activity and thus are useful in thecompositions of the invention, such as, but not limited to, e.g.,cleaning compositions (including, but not limited, to, e.g., detergentcleaning compositions) for cleaning a variety of surfaces and items,including, but not limited to, e.g., laundry, fabrics, and dishware, andin personal care applications and compositions as described elsewhereherein; such protease variants are also useful in fabric and home careproducts and compositions and in non-fabric and home care products andcompositions described elsewhere herein and in methods of the invention,including, but not limited, to, e.g., cleaning methods, methods forpersonal care, etc., described elsewhere herein.

Various terms set forth below are used to describe the variant:non-deleterious variants have a PI>0.05; deleterious variants have a PIless than or equal to 0.05; combinable variants are those for which thevariant has performance index values greater than or equal to 0.2 for atleast one property, and >0.05 for all properties. Combinable variantsare those that can be combined to deliver proteins with appropriateperformance indices for one or more desired properties. These data finduse in engineering any subtilisin/subtilase or protease. Even if thesubtilase or protease to be engineered has an amino acid different fromthat of subtilisin BPN′ at one or more particular positions, these datafind use in identifying amino acid substitutions that alter the desiredproperties by identifying the best choices for substitutions, includingsubstitutions of the BPN′ wild type amino acid.

Example 2 Construction of BPN′ Library and Cleaning Performance of BPN′Variants

a) Description of the BPN′-v3 Expression Cassette Used for LibraryConstruction

The BPN′-v3 (BPN′ protease containing G097A-G128A-Y217Q substitutions)expression cassette used for combinatorial library construction wasgenerated using the BPN′ expression cassette, which comprises theaprE-BPN′ hybrid leader sequence (i.e., signal sequence), BPN′ pro andBPN′ mature sequence from B. amyloliquefaciens. The DNA sequence isshown below as SEQ ID NO:1 and encodes the BPN′ precursor protein shownbelow as SEQ ID NO:168.

(SEQ ID NO: 1) GTGAGAAGCAAAAAATTGTGGATCAGTTTGCTGTTTGCTTTAGCGTTAATCTTTACGATGGCGTTCGGCAGCACATCCTCTGCCCAGGCGGCAGGGAAATCAAACGGGGAAAAGAAATATATTGTCGGGTTTAAACAGACAATGAGCACGATGAGCGCCGCTAAGAAGAAAGATGTCATTTCTGAAAAAGGCGGGAAAGTGCAAAAGCAATTCAAATATGTAGACGCAGCTTCAGCTACATTAAACGAAAAAGCTGTAAAAGAATTGAAAAAAGACCCGAGCGTCGCTTACGTTGAAGAA GATCACGTAGCACATGCGTACGCGCAGTCCGTGCCTTACGGCGTATCACAAATTAAAGCCCCTGCTCTGCACTCTCAAGGCTACACTGGATCAAATGTTAAAGTAGCGGTTATCGACAGCGGTATCGACTCGAGCCATCCAGATCTTAAAGTCGCTGGAGGGGCTTCTATGGTGCCGTCCGAAACAAACCCGTTTCAAGATAACAATTCTCATGGCACACACGTCGCAGGAACGGTTGCGGCGTTAAACAATTCTATTGGCGTGCTTGGTGTAGCCCCGTCTGCTTCGCTCTACGCCGTTAAAGTTCTTGGCGCAGACGGATCAGGCCAATACTCATGGATTATCAACGGCATCGAATGGGCCATCGCGAATAACATGGATGTAATCAACATGAGCCTGGGAGGACCAAGCGGCAGTGCGGCACTTAAAGCAGCAGTTGATAAAGCTGTTGCATCTGGTGTCGTCGTAGTAGCGGCAGCTGGGAATGAGGGAACATCCGGATCATCGAGTACCGTCGGTTATCCAGGCAAGTACCCTTCAGTGATTGCAGTGGGCGCTGTAGACTCTTCAAATCAACGTGCCTCTTTTTCCTCCGTGGGACCGGAGCTGGATGTCATGGCCCCTGGCGTTTCTATTCAATCGACGCTTCCAGGGAACAAGTATGGTGCGTATAACGGGACTTCCATGGCCTCGCCGCATGTAGCTGGGGCGGCCGCATTGATTCTTTCTAAGCACCCGAACTGGACAAACACTCAAGTCCGCAGCAGTTTAGAAAACACCACTACAAAACTTGGTGATTCTTTCTACTATGGAAAAGGGCTGATCAACGTACAGGCGGCAGCTCAG

In the nucleotide sequence of SEQ ID NO:1, the DNA sequence encoding themature protease is shown in bold, the nucleotide sequence encodingleader sequence (aprE-BPN′ hybrid leader sequence) is shown in standard(non-underlined) text, and the nucleotide sequence encoding the prosequence (BPN′) is underlined. In the amino acid sequence (aprE-BPN′hybrid leader sequence, BPN′ pro sequence, and BPN′ mature proteinsequence) of the BPN′ precursor protein set forth in SEQ ID NO:168, thebolded portion indicates the mature BPN′ subtilisin protease.

(SEQ ID NO: 168) VRSKKLWISLLFALALIFTMAFGSTSSAQAAGKSNGEKKYIVGFKQTMSTMSAAKKKDVISEKGGKVQKQFKYVDAASATLNEKAVKELKKDPSVAYVEEDHVAHAYAQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETNPFQDNNSHGTHVAGTVAALNNSIGVLGVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVQAAAQ

The amino acid sequence of the mature BPN′ subtilisin protease is shownas SEQ ID NO:2.

The nucleotide sequence of the mature BPN′-v3 gene is that of SEQ IDNO:3 (the signal sequence and propeptide sequence used in the BPN′-v3expression cassette is the same as that for BPN′ shown in SEQ ID NO:1).The protein sequence of the mature BPN′-v3 protease variant is that ofSEQ ID NO:4 (the signal sequence and propeptide sequence used in theBPN′-v3 expression cassette is the same as that for BPN′ shown in SEQ IDNO:168).

b) Construction of Combinatorial Library Using pHPLT-BPN′-v3 Plasmid

The pHPLT-BPN′-v3 plasmid (see FIG. 1) containing the BPN′-v3 expressioncassette described above served as template DNA for cloning to providevariants derived from BPN′-v3. The vector pHPLT (FIG. 4 in U.S. Pat. No.6,566,112) contains the B. licheniformis LAT promoter (“Plat”); asequence encoding the LAT signal peptide (“preLAT”). Additional plasmidelements from plasmid pUB110 disclosed in McKenzie et al., Plasmid15(2): 93-103 (1986): “ori-pUB” is the origin of replication frompUB110; “neo” is the neomycin/kanamycin resistance gene from pUB110;“Terminator” is the transcriptional terminator from B. licheniformisamylase.

A combinatorial DNA library was synthesized at DNA 2.0 and delivered asindividual ligation reactions. In some instances for efficienttransformation of B. subtilis, the DNA from the ligation reactionmixtures was amplified by rolling circle amplification (RCA) using theIllustra Templiphi kit (GE Healthcare). The reaction was performedaccording to the manufacturer's protocol. One microliter of ten-folddiluted amplified DNA was used to transform 50 μL of competent B.subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). Thetransformation mixture was shaken at 37° C. for 1 hour. Ten micro-literaliquots of the transformation mixture were plated on skim milk (1.6%)Luria agar plates supplemented with 10 μg/ml of neomycin (Teknova).

The transformants that formed halos on the skim milk plates were pickedinto microtiter plates containing 150 μl Luria broth (LB) mediumsupplemented with 10 μg/ml neomycin. Plates were grown overnight at 37°C. with 250-300 rpm shaking and 70-80% humidity using Enzyscreen lidsfor microtiter plates (Enzyscreen). Using a 96 pin replicating tool,(Enzyscreen) the overnight culture plate was used to inoculate a newmicrotiter plate containing 180 μl of MBD medium (a MOPS based definedmedium) with 2.5 μg/ml neomycin. MBD medium was prepared essentially asknown in the art (see Neidhardt et al., J. Bacteriol. 119:736-747[1974]), except that NH₄Cl, FeSO₄, and CaCl₂ were omitted from the basemedium, 3 mM K₂HPO₄ was used, and the base medium was supplemented with60 mM urea, and 100 ml of a solution made of 210 g/L glucose, and 350g/L maltodextrin. 1 g/L of BD Bacto Yeast Extract was added and the pHwas adjusted to 7.4 with KOH. The micronutrients were made up as a 100×stock solution containing in one liter, 400 mg FeSO₄.7H₂O, 100 mgMnSO₄.H₂O, 100 mg ZnSO₄.7H₂O, 50 mg CuCl₂.2H₂O, 100 mg CoCl₂.6H₂O, 100mg NaMoO₄.2H₂O, 100 mg Na₂B₄O₇.10H₂O, 10 ml of 1M CaCl₂, and 10 ml of0.5 M sodium citrate. The MBD medium containing microtiter plates weregrown for 64 hours at 37° C., 250-300 rpm, and 70-80% humidity usingEnzyscreen lids (Enzyscreen) for protease variant expression. The nextday, cultures were filtered through a micro-filter plate (0.22 um;Millipore) and the resulting filtrates containing protease variants wereused for biochemical analysis.

The protease variants were tested for cleaning performance using a BMImicroswatch assay in Detergent Composition 1 at 16° C. and pH 8 and BMImicroswatch assay in Detergent Composition 2 at 16° C. and pH 8. Proteincontent was determined using the TCA assay. Assays were performed asdescribed in Example 1 and Performance Indices were calculated relativeto BPN′-v3 (with a PI value of 1) as per Test Method 3.

The following BPN′ subtilisin protease variant was determined to have aPI value greater than 1.0, at least 1.1, or from greater than 1.0 toabout 5 relative to BPN′-v3 in a BMI microswatch cleaning assay inDetergent Composition 1 at pH 8 and 16° C.: BPN′ amino acid sequence(SEQ ID NO:2) comprising the set of amino acid substitutionsG097A-G128A-P210S-Y217Q, wherein amino acid positions of the variant arenumbered by correspondence with the sequence of SEQ ID NO:2. Suchvariant has a PI value of 1.1 relative to BPN′-v3 in this BMImicroswatch cleaning assay, and enhanced proteolytic activity comparedto BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay.The invention includes a protease variant having enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2) in this assay, the varianthaving an amino acid sequence comprising amino acid substitutionsG097A-G128A-P210S-Y217Q, wherein amino acid positions of the variant arenumbered by correspondence with the sequence of SEQ ID NO:2. Alsoincluded is a protease variant having enhanced proteolytic activitycompared to SEQ ID NO:2 in this assay, the variant comprising an aminoacid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2and comprising amino acid substitutions G097A-G128A-P210S-Y217Q, whereinamino acid positions of the variant are numbered by correspondence withamino acid positions of the SEQ ID NO:2 sequence. Also provided is asubtilisin protease variant having enhanced proteolytic activitycompared to BPN′ and/or a PI value greater than that of BPN′ (SEQ IDNO:2) and/or BPN′-v3 in a BMI microswatch cleaning assay, the variantcomprising an amino acid sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variantcomprises substitutions X097A-X128A-X210S-X217Q, wherein positions ofthe variant are numbered by correspondence with the sequence of SEQ IDNO:2, and optionally wherein the variant comprises at least onesubstitution selected from the group of G097A, G128A, P210S, and Y217Q.Such protease variant may be an isolated, recombinant, substantiallypure, or non-naturally occurring protease variant. Also included arecompositions, including, but not limited to, e.g., cleaningcompositions, comprising such protease variant and methods for cleaningutilizing such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal toabout 1.0 relative to BPN′-v3 (i.e., having a PI value approximatelyequivalent to that of BPN′-v3) in a BMI microswatch cleaning assay inDetergent Composition 1 at pH 8 and 16° C.: BPN′ amino acid sequence(SEQ ID NO:2) comprising at least one set of amino acid substitutionsselected from the group consisting of G097A-G128A-Y217Q,G097A-G128A-E156S-P210S-Y217Q, G097A-G128A-P210S-Y217Q-N218A,G097A-G128A-P210S-Y217Q-N218S, and G097A-Y104F-G128A-E156S-P210I-Y217Q,wherein amino acid positions of the variant are numbered bycorrespondence with the sequence of SEQ ID NO:2. Such variants haveenhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and agreater PI value than BPN′ in this assay. The invention includes aprotease variant having enhanced proteolytic activity compared to BPN′(SEQ ID NO:2) or a PI value of 1.0 relative to BPN′-v3 in this BMImicroswatch cleaning assay, the variant comprising an amino acidsequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2 andcomprising at least one set of amino acid substitutions selected fromsaid group above, wherein amino acid positions of the variant arenumbered by correspondence with amino acid positions of the SEQ ID NO:2sequence. Also provided is a subtilisin protease variant having enhancedproteolytic activity compared to BPN′ and/or a PI value of 1.0 comparedto BPN′-v3 in this BMI microswatch cleaning assay, the variantcomprising an amino acid sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variantcomprises at least one substitution selected from the group of X097A,X104F, X128A, X156A/S, X210I/S, X217Q, X218A/S, and optionally at leastone substitution selected from the group of G097A, Y104F, G128A,E156A/S, P210I/S, Y217Q, and N218A/S, wherein positions of the variantare numbered by correspondence with the sequence of SEQ ID NO:2. Alsoincluded are compositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′ variants were determined to have a PI value of about0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in DetergentComposition 1 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2)comprising at least one set of amino acid substitutions selected fromthe group consisting of G097A-G128A-E156A-P210S-Y217Q-N218S andG097A-G128A-Y217Q-N218A, wherein positions of the variant are numberedby correspondence with the sequence of SEQ ID NO:2. Such variants haveproteolytic activity. The invention includes a protease variant havingproteolytic activity, a PI value of 0.9 relative to BPN′-v3, and/orenhanced proteolytic activity compared to BPN′ in this assay, thevariant comprising an amino acid sequence having at least 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to thesequence of SEQ ID NO:2 and at least one set of amino acid substitutionsselected from said group above, wherein amino acid positions of thevariant are numbered by correspondence with positions of the SEQ ID NO:2sequence. Also included are compositions, including, but not limited to,e.g., cleaning compositions, comprising at least one such variant andmethods for cleaning utilizing at least one such variant as described ingreater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value ofgreater than 1.0 to about 5 relative to BPN′-v3 in a BMI microswatchcleaning assay in Detergent Composition 2 at pH 8 and 16° C.: BPN′ aminoacid sequence (SEQ ID NO:2) comprising at least one set of amino acidsubstitutions selected from the group consisting ofG097A-G128A-P210S-Y217Q-N218A and G097A-G128A-P210S-Y217Q, wherein aminoacid positions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. The invention includes a protease varianthaving enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2)and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v3 inthis BMI microswatch cleaning assay, the variant comprising an aminoacid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2and comprising at least one set of amino acid substitutions selectedfrom said group above, wherein amino acid positions of the variant arenumbered by correspondence with amino acid positions of the SEQ ID NO:2sequence. Such variants have enhanced proteolytic activity compared toBPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. Alsoprovided is a subtilisin protease variant having enhanced proteolyticactivity compared to BPN′ and/or a PI value of greater than 1 comparedto BPN′-v3 in this BMI microswatch cleaning assay, the variantcomprising an amino acid sequence having at least 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2, wherein thevariant comprises at least one substitution selected from the group ofX097A, X104F, X128A, X156A/S, X210I/S, X217Q, X218A/S, and optionally atleast one substitution selected from the group of G097A, Y104F, G128A,E156A/S, P210I/S, Y217Q, and N218A/S, wherein amino acid positions ofthe variant are numbered by correspondence with position of the sequenceof SEQ ID NO:2. Such variants have enhanced proteolytic activitycompared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in thisassay. Also included are compositions, including, but not limited to,e.g., cleaning compositions, comprising at least one such variant andmethods for cleaning utilizing at least one such variant as described ingreater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal toabout 1.0 relative to BPN′-v3 in a BMI microswatch cleaning assay inDetergent Composition 2 at pH 8 and 16° C.: BPN′ amino acid sequence(SEQ ID NO:2) comprising at least one set of amino acid substitutionsselected from the group consisting of G097A-G128A-Y217Q (i.e., BPN′-v3),G097A-G128A-E156S-P210S-Y217Q, and G097A-G128A-P210S-Y217Q-N218S,wherein positions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. Such variants have proteolytic activity andenhanced proteolytic activity compared to BPN′ in this assay. Theinvention includes a protease variant having proteolytic activity, PIvalue of 1.0 relative to BPN′-v3, and/or enhanced proteolytic activitycompared to BPN′ in this assay, the variant comprising an amino acidsequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2 and atleast one set of amino acid substitutions selected from said groupabove, wherein positions of the variant are numbered by correspondencewith positions of the SEQ ID NO:2 sequence. Also included arecompositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′ variants were determined to have a PI value of about0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in DetergentComposition 2 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2)comprising at least one set of amino acid substitutions selected fromthe group consisting of G097A-G128A-E156A-P210S-Y217Q-N218S,G097A-G128A-Y217Q-N218A, and G097A-Y104F-G128A-E156S-P210I-Y217Q,wherein positions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. Such variants have proteolytic activity. Theinvention includes a protease variant having proteolytic activity, a PIvalue of 0.9 relative to BPN′-v3, and/or enhanced proteolytic activitycompared to BPN′ in this assay, the variant comprising an amino acidsequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2 and atleast one set of amino acid substitutions selected from said groupabove, wherein amino acid positions of the variant are numbered bycorrespondence with positions of the SEQ ID NO:2 sequence. Also includedare compositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

Example 3 Generation of Combinatorial Libraries and Cleaning Performanceof Variants of BPN′-v3+S78N

a) Description of BPN′-v3+S78N Variant and Synthetic Gene SequencesDerived from this Variant

Gene Oracle synthesized and cloned eight genes into thepHPLT-BPN′-v3+S78N (BPN′-S78N-G97A-G128A-Y217Q) parent plasmid (see FIG.2). Some of these genes were used as templates (parents) to createcombinatorial libraries. The BPN′-v3+S78N variant was generated usingstandard molecular biology methods known in the art. The nucleotidesequence encoding the BPN′-v3+S78N variant is that of SEQ ID NO:7 andthe amino acid sequence of the BPN′-v3+S78N variant is that of SEQ IDNO:8. The nucleotide and protein sequences of genes GcM90-96, and GcM100are shown below.

Genes Nucleotide Sequence Protein Sequence GcM90 SEQ ID NO: 9 SEQ ID NO:10 GcM91 SEQ ID NO: 11 SEQ ID NO: 12 GcM92 SEQ ID NO: 13 SEQ ID NO: 14GcM93 SEQ ID NO: 15 SEQ ID NO: 16 GcM94 SEQ ID NO: 17 SEQ ID NO: 18GcM95 SEQ ID NO: 19 SEQ ID NO: 20 GcM96 SEQ ID NO: 21 SEQ ID NO: 22GcM100 SEQ ID NO: 23 SEQ ID NO: 24

b) Construction of Combinatorial Libraries CG1-CG5 and CG8 Using theSynthetic Genes GcM90-94 and GcM100

TABLE 3-1List of Possible Substitutions Introduced and Primers Used for theConstruction of Combinatorial Libraries CG1-CG5 and CG8 SynthesizedGenes (template or Library Substitutions Primer parent) Name IntroducedName Primer Sequence GcM90 CG1 G53S 1 S53 f /5Phos/CTTCTATGGTGCCGTCCGAAACAAACCCGTTTCAAG (SEQ ID NO: 25) A68V 1 V68 f/5Phos/TCATGGCACACACGTCGCAGG AACGGTTGCGGCG (SEQ ID NO: 26) A102G1 G102 f /5Phos/AGCAGACGGATCAGGCCAAT ACTCATGGATTATCAAC (SEQ ID NO: 27)T129P 1 P129 f /5Phos/TGAGCCTGGGAGCACCAAGC GGCAGTGCGGCACTTAAAG (SEQ IDNO: 28) T185Q 1 Q185 f /5Phos/TAGACTCTTCAAATCAACGTGCCTCTTTTTCCTCCGTG (SEQ ID NO: 29) GcM91 CG2 V59Q 2 Q59 f/5Phos/GAAACAAACCCGTTTCAAGAT AACAATTCTCATG (SEQ ID NO: 30) V108I2 I108 f /5Phos/ATACTCATGGATTATCAACGG CATCGAATGGGCCATC (SEQ ID NO: 31)Q147V 2 V147 f /5Phos/TGTTGCATCTGGTGTCGTCGT AGTAGCGGCAGCTGG (SEQ IDNO: 32) A211G 2 G211 f /5Phos/ATCGACGCTTCCAGGGAACAAGTATGGTGCGCAAAAC (SEQ ID NO: 33) Q252N 2 N252 f/5Phos/CAGCAGTTTAGAAAACACCA CTACAAAACTTGGTG (SEQ ID NO: 34) GcM92 CG3A61N 3 N61 f /5Phos/CAAACCCGTTTCAAGATAACA ATTCTCATGGCACACAC (SEQ IDNO: 35) E87S 3 S87 f /5Phos/TTGGTGTAGCCCCGTCTGCTTCGCTCTACGCCGTTAAAG (SEQ ID NO: 36) I124M 3 M124 f/5Phos/TGGATGTAATCAACATGAGCC TGGGAGCACCAAGCG (SEQ ID NO: 37) P161S3 S161 f /5Phos/AGGGAACATCCGGATCATCG AGTACCGTCGGTTATCCAG (SEQ ID NO: 38)A224S 3 S224 f /5Phos/GACTTCCATGGCCTCGCCGCATGTAGCTGGGGCGGC (SEQ ID NO: 39) GcM93 CG4 Q62N 4 N62 f/5Phos/GTTTCAAGATAACAATTCTCA TGGCACACACGTCGC (SEQ ID NO: 40) N100G4 G100 f /5Phos/GTTCTTGCAGCAGACGGATCA GGCCAATACTCATG (SEQ ID NO: 41)A125S 4 S125 f /5Phos/ATGTAATCAACATGAGCCTGG GAGCACCAAGCGGCAG (SEQ IDNO: 42) D159S 4 S159 f /5Phos/GGAATGAGGGAACATCCGGATCATCGAGTACCGTCGG (SEQ ID NO: 43) S240N 4 N240 f/5Phos/CTTTCTAAGCACCCGAACTGG ACAAACACTCAAGTCCG (SEQ ID NO: 44) GcM94 CG5T63S 5 S63 f /5Phos/TCAAGATAACAATTCTCATGGCACACACGTCGCAGG (SEQ ID NO: 45) A101S 5 S101 f/5Phos/TGCAGCAGACGGATCAGGCC AATACTCATGGATTATC (SEQ ID NO: 46) V126L5 L126 f /5Phos/AATCAACATGAGCCTGGGAG CACCAAGCGGCAGTG (SEQ ID NO: 47)T183S 5 S183 f /5Phos/CGCTGTAGACTCTTCAAATCA ACGTGCCTCTTTTTCC (SEQ IDNO: 48) N244T 5 T244 f /5Phos/GAACTGGACAAACACTCAAGTCCGCAGCAGTTTAG (SEQ ID NO: 49) GcM100 CG8 A68V 1 V68 f/5Phos/TCATGGCACACACGTCGCAGG AACGGTTGCGGCG (SEQ ID NO: 50) A102G1 G102 f /5Phos/AGCAGACGGATCAGGCCAAT ACTCATGGATTATCAAC (SEQ ID NO: 51)A211G 2 G211 f /5Phos/ATCGACGCTTCCAGGGAACA AGTATGGTGCGCAAAAC (SEQ IDNO: 52) A125S 4 S125 f /5Phos/ATGTAATCAACATGAGCCTGGGAGCACCAAGCGGCAG (SEQ ID NO: 53)

Each synthesized gene was built into thepHPLT-BPN′-S78N-G97A-G128A-Y217Q parent molecule. Resulting plasmidscontaining the six synthesized genes GcM90-94, and GcM100 served astemplates to make combinatorial libraries at the respective positions(Table 3-1). Two additional genes, GcM95 and GcM96, were alsosynthesized for analysis, but did not serve as parental DNA forlibraries. These genes each have nine mutations on top of thepHPLT-BPN′-S78N-G97A-G128A-Y217Q parent molecule.

The parent plasmids (template DNA) containing the synthetic genesGcM90-94, and GcM100 were methylated were methylated using twomicrograms of DNA and methylase (NEB), according to the NEB protocol.Methylated DNA was then purified using DNA Clean and Concentrator kit(Zymo Research). Combinatorial libraries CG1-5 and CG8 were made using aQUIKCHANGE® Multi Site-Directed Mutagenesis kit (“QCMS kit”; Stratagene)following the manufacturer's protocol (see Table 3-1 for respectivetemplate and primer combinations), with the exception of libraries CG3and CG4, which used 86.5 ng of each primer in place of the 50 ngsuggested in the protocol. All primers used for introducing the desiredsubstitutions in each library are listed in Table 3-1. They weresynthesized and provided by Integrated DNA Technologies. After the QCMSreactions were completed for each library, the template DNA was digestedby the addition of 0.5-1 μl DpnI (from the QCMS kit) and incubated at37° C. for 1-4 hours, followed by another addition of 0.5-1 μl DpnI andanother incubation at 37° C. for 1-4 hours. For efficient transformationof B. subtilis, DNA from the QCMS reaction mixtures were amplifiedbefore transformation and transformants grown as described in Example 2.

Additional variants of BPN′-v3+S78N were produced by DNA2.0. Thefollowing substitutions were introduced individually into theBPN′-v3+S78N parent molecule: Q59G, N62Q, V68A, S89Y, A92G, I108V,I115V, M124T, P129L, A138T, V147L, S161P, Y167A, P172V, G211T, L267V,and A273S.

All of the combinatorial library variants described above and thevariants synthesized at DNA2.0 were tested for cleaning performanceusing a BMI microswatch assay of Test Method 3 in Detergent Composition1 at 16° C. and pH 8 and BMI microswatch assay in Detergent Composition2 at 16° C. and pH 8. Protein content was determined using the TCAassay. Assays were performed as described in Example 1 and PerformanceIndices were calculated relative to the enzyme of SEQ ID NO:4 (with a PIvalue of 1.0).

The following BPN′ variants were determined to have a PI value greaterthan 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, atleast 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, atleast 2, from greater than 1.0 to about 10, from greater than 1.0 toabout 8, or from greater than 1.0 to about 5 relative to the enzyme ofSEQ ID NO:4 in a BMI microswatch cleaning assay in Detergent Composition1 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprisingat least one set of amino acid substitutions selected from the groupconsisting of S063T-S078N-G097A-S101A-G128A-S183T-Y217Q-T244N,N061A-S078N-G097A-G128A-Y217Q-S224A,S053G-S078N-G097A-G128A-P129T-Q185T-Y217Q,S063T-S078N-G097A-S101A-G128A-S183T-Y217Q,S063T-S078N-G097A-S101A-G128A-Y217Q,S063T-S078N-G097A-S101A-G128A-Y217Q-T244I, andS078N-G097A-G128A-P129T-Y217Q, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have enhanced proteolytic activity compared to BPN′ (SEQID NO:2) and a greater PI value than BPN′ and BPN′-v3 in this assay. Theinvention includes a protease variant having enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2) and/or a PI value of greaterthan 1 to about 5 relative to BPN′-v3 in this BMI microswatch cleaningassay, the variant comprising an amino acid sequence having at least85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQID NO:2 and comprising at least one set of amino acid substitutionsselected from said group above, wherein amino acid positions of thevariant are numbered by correspondence with amino acid positions of theSEQ ID NO:2 sequence. Also included are compositions, including, but notlimited to, e.g., cleaning compositions, comprising at least one suchvariant and methods for cleaning utilizing at least one such variant asdescribed in greater detail elsewhere herein.

Also provided is a subtilisin protease variant having enhancedproteolytic activity compared to BPN′ and/or a PI value of greater than1.0 to about 5 relative to BPN′-v3 in this BMI microswatch cleaningassay, the variant comprising an amino acid sequence having at least85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ IDNO:2, wherein the variant comprises at least one substitution selectedfrom the group of X040E, X053G, X059V, X061A, X062H/Q, X068A, X078N,X087E, X101A, X102A, X108V, X124I, X125A, X126V, X129T, X147Q, X159D,X183T, X185T, X211A, X224A, X244I/N, X252Q, and X274D, and optionally atleast one substitution selected from the group of X040E, X053G, X059V,X061A, X062H/Q, X068A, X078N, X087E, X101A, X102A, X108V, M124I, S125A,L126V, P129T, V147Q, S159D, S183T, Q185T, G211A, S224A, T244I/N, N252Q,and A274D, wherein amino acid positions of the variant are numbered bycorrespondence with positions of the sequence of SEQ ID NO:2. Alsoincluded are compositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal toabout 1.0 relative to the enzyme of SEQ ID NO:4 (alternatively referredto as BPN′-v3) in a BMI microswatch cleaning assay in DetergentComposition 1 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2)comprising at least one set of amino acid substitutions selected fromthe group consisting of G097A-G128A-Y217Q,N061A-S078N-S087E-G097A-G128A-Y217Q-S224A,Q059V-S078N-G097A-G128A-G211A-Y217Q,Q059V-S078N-G097A-G128A-V147Q-Y217Q, Q059V-S078N-G097A-G128A-Y217Q,Q059V-S078N-G097A-I108V-G128A-Y217Q-N252Q,S053G-S078N-G097A-G128A-P129T-Y217Q, S078N-G097A-G128A-G211A-Y217Q,S078N-G097A-G128A-Q185T-Y217Q, S078N-G097A-G128A-V147Q-Y217Q,S078N-G097A-G128A-Y217Q, S078N-G097A-G128A-Y217Q-S224A, andS078N-G097A-G128A-Y217Q-S224A-A274D, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have enhanced proteolytic activity compared to BPN′ (SEQID NO:2) and a greater PI value than BPN′ in this assay. The inventionincludes a protease variant having enhanced proteolytic activitycompared to BPN′ (SEQ ID NO:2) and/or a PI value of 1.0 relative toBPN′-v3 in this BMI microswatch cleaning assay, the variant comprisingan amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQID NO:2 and comprising at least one set of amino acid substitutionsselected from said group above, wherein amino acid positions of thevariant are numbered by correspondence with amino acid positions of theSEQ ID NO:2 sequence. Also included are compositions, including, but notlimited to, e.g., cleaning compositions, comprising at least one suchvariant and methods for cleaning utilizing at least one such variant asdescribed in greater detail elsewhere herein.

The following BPN′ variant was determined to have a PI value of about0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in DetergentComposition 1 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2)comprising the set of amino acid substitutionsS078N-G097A-I108V-G128A-V147Q-Y217Q, wherein positions of the variantare numbered by correspondence with the sequence of SEQ ID NO:2. Suchvariants have proteolytic activity. The invention includes a proteasevariant having proteolytic activity, a PI value of 0.9 relative toBPN′-v3, and/or enhanced proteolytic activity compared to BPN′ in thisassay, the variant comprising an amino acid sequence having at least60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or99% identity to SEQ ID NO:2 and comprising said set of amino acidsubstitutions above, wherein amino acid positions of the variant arenumbered by correspondence with positions of the SEQ ID NO:2 sequence.Also included are compositions, including, but not limited to, e.g.,cleaning compositions, comprising at least one such variant and methodsfor cleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′ variants were determined to a PI value equal to orgreater than 0.5 and less than 0.9 relative to BPN′-v3 in a BMImicroswatch cleaning assay in Detergent Composition 1 at pH 8 and 16°C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one setof amino acid substitutions selected from the group consisting ofQ059V-S078N-G097A-I108V-G128A-V147Q-G211A-Y217Q-N252Q,S078N-G097A-I108V-G128A-V147Q-G211A-Y217Q,S078N-G097A-I108V-G128A-V147Q-G211A-Y217Q-N252Q,S078N-G097A-I108V-G128A-V147Q-Y217Q-N252Q, andS078N-S087E-G097A-M124I-G128A-Y217Q-S224A, wherein amino acid positionsof the variant are numbered by correspondence with positions of the SEQID NO:2 sequence. Such variants have proteolytic activity. The inventionincludes a protease variant having proteolytic activity, the variantcomprising an amino acid sequence having at least 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ IDNO:2 and at least one set of amino acid substitutions selected from saidgroup above, wherein positions of the variant are numbered bycorrespondence with positions of the SEQ ID NO:2 sequence. Also includedare compositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′ variants were determined to have a PI value greaterthan 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, atleast 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, atleast 2, from greater than 1.0 to about 10, from greater than 1.0 toabout 8, or from greater than 1.0 to about 5 relative to BPN′-v3 in aBMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least oneset of amino acid substitutions selected from the group consisting ofS063T-S078N-G097A-S101A-G128A-S183T-Y217Q,S063T-S078N-G097A-S101A-G128A-S183T-Y217Q-T244N,S063T-S078N-G097A-S101A-G128A-Y217Q, andS063T-S078N-G097A-S101A-G128A-Y217Q-T244I, wherein amino acid positionsof the variant are numbered by correspondence with the sequence of SEQID NO:2. Such variants have enhanced proteolytic activity compared toBPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. Theinvention includes a protease variant having enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2) and/or a PI value of greaterthan 1.0 to about 5 relative to BPN′-v3 in this BMI microswatch cleaningassay, the variant comprising an amino acid sequence having at least60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least oneset of amino acid substitutions selected from said group above, whereinamino acid positions of the variant are numbered by correspondence withamino acid positions of the SEQ ID NO:2 sequence. Also included arecompositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal toabout 1.0 relative to BPN′-v3 in a BMI microswatch cleaning assay inDetergent Composition 2 at pH 8 and 16° C.: BPN′ amino acid sequence(SEQ ID NO:2) comprising at least one set of amino acid substitutionsselected from the group consisting of G097A-G128A-Y217Q,N061A-S078N-G097A-G128A-Y217Q-S224A,N061A-S078N-S087E-G097A-G128A-Y217Q-S224A,Q059V-S078N-G097A-G128A-G211A-Y217Q,Q059V-S078N-G097A-G128A-V147Q-Y217Q, Q059V-S078N-G097A-G128A-Y217Q,Q059V-S078N-G097A-I108V-G128A-Y217Q-N252Q,S053G-S078N-G097A-G128A-P129T-Q185T-Y217Q,S053G-S078N-G097A-G128A-P129T-Y217Q, S078N-G097A-G128A-G211A-Y217Q,S078N-G097A-G128A-P129T-Y217Q, S078N-G097A-G128A-Q185T-Y217Q,S078N-G097A-G128A-Y217Q, S078N-G097A-G128A-Y217Q-S224A, andS078N-G097A-G128A-Y217Q-S224A-A274D, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have enhanced proteolytic activity compared to BPN′ (SEQID NO:2) and a greater PI value than BPN′ in this assay. The inventionincludes a protease variant having enhanced proteolytic activitycompared to BPN′ (SEQ ID NO:2) and/or a PI value of about 1 relative toBPN′-v3 in this BMI microswatch cleaning assay, the variant comprisingan amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 andcomprising at least one set of amino acid substitutions selected fromsaid group above, wherein amino acid positions of the variant arenumbered by correspondence with amino acid positions of the SEQ ID NO:2sequence. Also included are compositions, including, but not limited to,e.g., cleaning compositions, comprising at least one such variant andmethods for cleaning utilizing at least one such variant as described ingreater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value of about0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in DetergentComposition 2 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2)comprising at least one set of amino acid substitutions selected fromthe group consisting of S078N-G097A-G128A-V147Q-Y217Q andS078N-G097A-I108V-G128A-V147Q-Y217Q, wherein positions of the variantare numbered by correspondence with the sequence of SEQ ID NO:2. Suchvariants have proteolytic activity. The invention includes a proteasevariant having proteolytic activity, a PI value of 0.9 relative toBPN′-v3, and/or an enhanced proteolytic activity compared to BPN′ inthis assay, the variant comprising an amino acid sequence having atleast 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%or 99% identity to SEQ ID NO:2 and at least one set of amino acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with positionsof the SEQ ID NO:2 sequence. Also included are compositions, includingcleaning compositions, comprising at least one such variant and methodsfor cleaning an item or surface in need of cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to a PI value equal to orgreater than 0.5 and less than 0.9 relative to BPN′-v3 in a BMImicroswatch cleaning assay in Detergent Composition 2 at pH 8 and 16°C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one setof amino acid substitutions selected from the group consisting ofQ059V-S078N-G097A-I108V-G128A-V147Q-G211A-Y217Q-N252Q,S078N-G097A-I108V-G128A-V147Q-G211A-Y217Q,S078N-G097A-I108V-G128A-V147Q-G211A-Y217Q-N252Q,S078N-G097A-I108V-G128A-V147Q-Y217Q-N252Q, andS078N-S087E-G097A-M124I-G128A-Y217Q-S224A, wherein amino acid positionsof the variant are numbered by correspondence with positions of the SEQID NO:2 sequence. Such variants have proteolytic activity. The inventionincludes a protease variant having proteolytic activity, the variantcomprising an amino acid sequence having at least 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ IDNO:2 and at least one set of amino acid substitutions selected from saidgroup above, wherein positions of the variant are numbered bycorrespondence with positions of the SEQ ID NO:2 sequence. Also includedare compositions, including cleaning compositions, comprising at leastone such variant and methods for cleaning an item or surface in need ofcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

Example 4 Generation and Cleaning Performance of BPN′ Variants

Generation of BPN′ Variants LC1-LC4 via QUIKCHANGE® Multi Site-DirectedMutagenesis

BPN′ variants were constructed from different parental plasmids usingQUIKCHANGE® Multi Site-Directed Mutagenesis kits. The parental plasmids(Table 4-1) were methylated using a NEB Dam Methylase Kit in a reactioncontaining 77.5 μL H₂O+10 μL Buffer 10×+0.25 μL SAM+2 uL DAMmethylase+10 uL miniprep DNA (˜150 ng/μL) at 37° C. overnight. Themethylated plasmid DNA was purified using a QIAGEN® PCR purificationkit. QUIKCHANGE® Multi Site-Directed Mutagenesis reactions were set upfor each of the DNA templates in a reaction mix containing 2.5 μL Buffer5×+0.5 μL primer 1 (25 μM)+0.5 μL primer 2 (25 μM)+1 μL dNTP's+1 μLenzyme blend+18 μL H₂O+1.5 μL DNA. The PCR program used was: 95° C. for1 min; (95° C. for 1 min, 53° C. for 1 min, 65° C. for 9:39 min)×29cycles; 65° C. for 10 min, 4° C. hold. Primer sequences are shown inTable 4-2. In all reactions, PCR was performed using a MJ ResearchPTC-200 Peltier thermal cycler. Parental DNA from the PCR samples wasremoved by addition of 1 μL of DpnI to QUIKCHANGE® Multi Site-DirectedMutagenesis reactions at 37° C. overnight. To increase transformationfrequency, the DpnI-digested reactions were amplified using rollingcircle amplification (RCA) using the Illustra TempliPhi kit according tothe manufacturer's protocol. B. subtilis cells (ΔaprE, ΔnprE,amyE::xylRPxylAcomK-phleo) were transformed with 1 μL each of the RCAreaction and the transformed cells were plated onto LA+1.6% skim milkplates containing 10 ppm neomycin and incubated at 37° C. overnight.Colonies from overnight growth were selected to perform colony PCR forsequencing using “puReTaq Ready-To-Go PCR Beads” (Amersham) The PCR andsequencing primers used were pHPLT F1 (/5PHOS/TACATATGAGTTATGCAGTTTG(SEQ ID NO:54)) and pHPLT seq R1 (/5PHOS/TTATCCTTTACCTTGTCTC (SEQ IDNO:55)). Clones with appropriate sequences were frozen. BPN′ variantproteins were produced by growing B. subtilis transformants in 96 wellmicrotiter plates at 37° C. for 68 hours in a MOPS based mediumcontaining urea as described in Example 2.

TABLE 4-1 Parental Plasmids and Primers Used for Generation of BPN′Variants LC1-LC4 Mutations Parental Plasmid Introduced Primers UsedBPN′-G097A-G128A-Y217Q-S024G- A128S A128Sf, A128Sr N025G-N061P-S101N(termed LC1) BPN′-G097A-G128A-Y217Q-S053G- A128S A128Sf, A128SrN061P-S101N-V203Y (termed LC2) BPN′-G097A-G128A-Y217Q-S024G- A128SA128Sf, A128Sr N025G-S053G-T055P-N061P-S101N- V203Y (termed LC3)BPN′-G097A-G128A-Y217Q-S024G- P55T P55Tf, P55TrN025G-S053G-T055P-N061P-S101N- V203Y (termed LC4)

TABLE 4-2 Sequences of Primers Used for QUIKCHANGE ® MultiSite-Directed Mutagenesis Reactions to Make BPN′ variants LC1-LC4 PrimerName Primer Sequence (5′ to 3′) A128Sf/5Phos/CAACATGAGCCTGGGATCACCAAGCGGCAGTGCGG (SEQ ID NO: 56) A128Sr/5Phos/CCGCACTGCCGCTTGGTGATCCCAGGCTCATGTTG (SEQ ID NO: 57) P55Tf/5Phos/CTATGGTGCCGGGCGAAACAAACCCGTTTCAAGATCCG (SEQ ID NO: 58) P55Tr/5Phos/CGGATCTTGAAACGGGTTTGTTTCGCCCGGCACCATAG (SEQ ID NO: 59)Generation of Additional BPN′ Variants LC5-LC37

An additional 33 BPN′ variants termed successively LC5 through LC37 wereproduced by DNA 2.0 using the BPN′ nucleic acid as the parent genecontained in the expression plasmid pHPLT-BPN′ partial opt (see FIG. 3).LC5 through LC37 BPN′ variants are as follows, respectively:BPN′-P52L-V68A-G97A-I111V, BPN′-I111V-M124V-Y167A-Y217Q,BPN′-Y104N-G128A-Y217Q, BPN′-M124V-Y167A-Y217Q, BPN′-I111V-M124V-Y217Q,BPN′-P52L-V68A-G97A, BPN′-G97A-I111V-M124V, BPN′-V68A-A92G-G97A,BPN′-G97A-I111V-M124V-Y167A-Y217Q, BPN′-P52L-V68A-I111V-Y217Q,BPN′-P52L-V68A-I111V, BPN′-V68A-A92G-I111V,BPN′-P52L-V68A-G97A-I111V-Y217Q, BPN′-V68A-G97A-I111V,BPN′-G97A-I111V-Y217Q, BPN′-G97A-I111V-M124V-Y167A,BPN′-S89Y-I111V-M124V, BPN′-V68A-S89Y-I111V, BPN′-V68A-A92G-Y217Q,BPN′-I111V-Y167A-Y217Q, BPN′-G97A-I111V-Y167A-Y217Q,BPN′-G97A-I111V-M124V-Y217Q, BPN′-V68A-I111V-Y167A-Y217Q,BPN′-I111V-G128A-Y217Q, BPN′-G97A-M124V-Y217Q, BPN′-V68A-Y167A-Y217Q,BPN′-I111V-M124V-Y167A, BPN′-N62Q-G97A-I111V,BPN′-G97A-M124V-Y167A-Y217Q, BPN′-G97A-L126A-Y217Q,BPN′-V68A-I111V-Y217Q, BPN′-S89Y-M124V-Y217Q, and BPN′-L96T-G97A-Y217Q.Plasmid pHPLT-BPN′ partial opt was also created by DNA 2.0.

Transformants were picked into microtiter plates and grown as describedin Example 2. The variants were assayed for cleaning performance using aBMI microswatch assay in Detergent Composition 2 at 16° C. and pH 8.Protein content was determined using the TCA assay. The assays wereperformed as described in Example 1 and the Performance Indices werecalculated relative to BPN′-v3 (with a PI value of 1.0).

The following BPN′ variants were determined to have a PI value greaterthan 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, atleast 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, atleast 2, from greater than 1.0 to about 10, from greater than 1.0 toabout 8, or from greater than 1.0 to about 5 relative to BPN′-v3 in aBMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least oneset of amino acid substitutions selected from the group consisting ofG097A-I111V-M124V-Y217Q, G097A-I111V-Y167A-Y217Q,S024G-N025G-N061P-G097A-S101N-G128S-Y217Q,S024G-N025G-S053G-N061P-G097A-S101N-G128A-V203Y-Y217Q,S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128S-V203Y-Y217Q, andV068A-A092G-Y217Q, wherein amino acid positions of the variant arenumbered by correspondence with the sequence of SEQ ID NO:2. Suchvariants have enhanced proteolytic activity compared to BPN′ (SEQ IDNO:2) and a greater PI value than BPN′ in this assay. The inventionincludes a protease variant having enhanced proteolytic activitycompared to BPN′ (SEQ ID NO:2) and/or a PI value of greater than 1.0 toabout 5 relative to BPN′-v3 in this BMI microswatch cleaning assay, thevariant comprising an amino acid sequence having at least 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity toSEQ ID NO:2 and comprising at least one set of amino acid substitutionsselected from said group above, wherein amino acid positions of thevariant are numbered by correspondence with amino acid positions of theSEQ ID NO:2 sequence. Also provided is a subtilisin protease varianthaving enhanced proteolytic activity compared to BPN′ and/or a PI valueof greater than 1.0 compared to BPN′-v3 in this BMI microswatch cleaningassay, the variant comprising an amino acid sequence having at least85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ IDNO:2, wherein the variant comprises at least one substitution selectedfrom the group of X024G, X025G, X052L, X053G, X055P, X061P, X062Q,X068A, X089Y, X092G, X096T, X097A, X101N, X104N, X111V, X124V, X126A,X128A/S, X167A, X203Y, and X217Q, and optionally at least onesubstitution selected from the group of S024G, N025G, P052L, S053G,T055P, N061P, N062Q, V068A, S089Y, A092G, L096T, G097A, S101N, Y104N,I111V, M124V, L126A, G128A/S, Y167A, V203Y, Y217Q, wherein amino acidpositions of the variant are numbered by correspondence with positionsof the sequence of SEQ ID NO:2. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal toabout 1.0 relative to BPN′-v3 in a BMI microswatch cleaning assay inDetergent Composition 2 at pH 8 and 16° C.: BPN′ amino acid sequence(SEQ ID NO:2) comprising at least one set of amino acid substitutionsselected from the group consisting of G097A-G128A-Y217Q,G097A-G128S-Y217Q, G097A-I111V-Y217Q, I111V-G128A-Y217Q,I111V-M124V-Y167A, I111V-M124V-Y217Q, L096T-G097A-Y217Q,N062Q-G097A-I111V, S053G-N061P-G097A-S101N-G128S-V203Y-Y217Q,S089Y-M124V-Y217Q, and V068A-I111V-Y217Q, wherein amino acid positionsof the variant are numbered by correspondence with the sequence of SEQID NO:2. The invention includes a protease variant having enhancedproteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of1.0 relative to BPN′-v3 in this BMI microswatch cleaning assay, thevariant comprising an amino acid sequence having at least 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to thesequence of SEQ ID NO:2 and comprising at least one set of amino acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Such variants have enhancedproteolytic activity compared to BPN′ (SEQ ID NO:2), a greater PI valuethan BPN′, and a PI value of 1.0 compared to BPN′-v3 in this assay. Alsoincluded are compositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′ variants were determined to have a PI value of about0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in DetergentComposition 2 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2)comprising at least one set of amino acid substitutions selected fromthe group consisting of G097A-I111V-M124V, G097A-L126A-Y217Q,G097A-M124V-Y217Q, I111V-Y167A-Y217Q, M124V-Y167A-Y217Q,P052L-V068A-G097A, S089Y-I111V-M124V, V068A-A092G-G097A,V068A-A092G-I111V, V068A-G097A-I111V, V068A-S089Y-I111V, andY104N-G128A-Y217Q, wherein positions of the variant are numbered bycorrespondence with the sequence of SEQ ID NO:2. Such variants haveproteolytic activity. The invention includes a protease variant havingproteolytic activity, a PI value of 0.9 relative to BPN′-v3, and/or anenhanced proteolytic activity compared to BPN′ in this assay, thevariant comprising an amino acid sequence having at least 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity toSEQ ID NO:2 and at least one set of amino acid substitutions selectedfrom said group above, wherein amino acid positions of the variant arenumbered by correspondence with positions of the SEQ ID NO:2 sequence.Also included are compositions, including, but not limited to, e.g.,cleaning compositions, comprising at least one such variant and methodsfor cleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′ variants were determined to a PI value equal to orgreater than 0.5 and less than 0.9 relative to BPN′-v3 in a BMImicroswatch cleaning assay in Detergent Composition 2 at pH 8 and 16°C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one setof amino acid substitutions selected from the group consisting ofG097A-M124V-Y167A-Y217Q, V068A-Y167A-Y217Q, G097A-I111V-M124V-Y167A,I111V-M124V-Y167A-Y217Q, V068A-I111V-Y167A-Y217Q,G097A-I111V-M124V-Y167A-Y217Q, P052L-V068A-I111V, wherein positions ofthe variant are numbered by correspondence with the sequence of SEQ IDNO:2. Such variants have proteolytic activity. The invention includes aprotease variant having proteolytic activity, the variant comprising anamino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and atleast one set of amino acid substitutions selected from said groupabove, wherein positions of the variant are numbered by correspondencewith positions of the SEQ ID NO:2 sequence. Such variants haveproteolytic activity. Also included are compositions, including, but notlimited to, e.g., cleaning compositions, comprising at least one suchvariant and methods for cleaning utilizing at least one such variant asdescribed in greater detail elsewhere herein.

Example 5 Cleaning Performance of BPN′ Variants

Variants based on parent BPN′ were made by DNA 2.0. The variants weregrown as described in Example 2 and tested for cleaning performance onBMI microswatch assay in Detergent Composition 1 at 16° C. and pH 8, BMImicroswatch assay in Detergent Composition 4 at 16° C. and pH 8, and eggmicroswatch assay in Detergent Composition 4 at 16° C. and pH 8. Theprotein content was determined using the TCA assay. The assays wereperformed as described in Example 1 and the Performance Indices werecalculated relative to BPN′-v3 (with a PI value of 1.0).

The following BPN′ variants were determined to have a PI value greaterthan 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, atleast 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, atleast 2, from greater than 1.0 to about 10, from greater than 1.0 toabout 8, or from greater than 1.0 to about 5 relative to BPN′-v3 in aBMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least oneset of amino acid substitutions selected from the group consisting ofN061P-G097A-S101N-G128A-P210S-Y217Q,S024G-N025G-S053G-N061P-G097A-S101N-G128A-P210S-Y217Q,S024G-N025G-S053G-N061P-G097A-S101N-G128S-Y217Q,S024G-N025G-S053G-N061P-S078N-G097A-S101N-I111V-G128S-Y217Q, whereinamino acid positions of the variant are numbered by correspondence withthe sequence of SEQ ID NO:2. Such variants have enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′in this assay. The invention includes a protease variant having enhancedproteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value ofgreater than 1.0 to about 5 relative to BPN′-v3 in this BMI microswatchcleaning assay, the variant comprising an amino acid sequence having atleast 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%or 99% identity to SEQ ID NO:2 and comprising at least one set of aminoacid substitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal toabout 1.0 relative to BPN′-v3 in a BMI microswatch cleaning assay inDetergent Composition 4 at pH 8 and 16° C.: BPN′ amino acid sequence(SEQ ID NO:2) comprising at least one set of amino acid substitutionsselected from the group consisting of G097A-G128A-Y217Q,G097A-G128A-P210S-Y217Q, G097A-G128S-P201S-Y217Q,G097A-I111V-M124I-Y217Q, G097A-I111V-M124V-P201S-Y217Q,G097A-N123Q-P210S-Y217Q, G097A-N123Q-Y217Q,N061P-G097A-G128A-P210S-Y217Q, N061P-G097A-G128S-Y217Q,N061P-G097A-I111V-M124V-Y217Q, N061P-G097A-N123Q-Y217Q,N061P-G097A-S101N-I111V-M124V-Y217Q, N061P-G097A-S101N-N123Q-Y217Q,N061P-G102A-P129S-Y217Q, N061P-N062Q-G097A-G100N-S101N-Y217Q,N061P-N062Q-G097A-G100N-Y217Q, N061P-N062Q-G097A-G100Q-P210S-Y217Q,N061P-N062Q-G097A-I111V-Y217Q, N061P-N062Q-G097A-S101N-I111V-Y217Q,N061P-S078N-G097A-I111V-M124I-Y217Q,N061P-S078N-G102A-I111V-P129S-Y217Q, N062Q-G097A-I111V-P201S-Y217Q,N062Q-G097A-I111V-Y217Q, N062Q-S078N-G097A-I111V-Y217Q,S024G-N025G-N061P-G097A-S101N-G128A-P210S-Y217Q,S024G-N025G-S053G-N061P-G097A-S101N-I111V-M124V-Y217Q,S024G-N025G-S053G-N061P-G097A-S101N-N123Q-Y217Q,S024G-N025G-S053G-N061P-N062Q-G097A-G100N-S101N-Y217Q,S024G-N025G-S053G-N061P-N062Q-G097A-S101N-I111V-Y217Q,S024G-N025G-S053G-N061P-S101N-G102A-P129S-Y217Q,S053G-N061P-G097A-G128S-Y217Q, S053G-N061P-G097A-M124I-Y217Q,S053G-N061P-G097A-S101N-I111V-M124V-Y217Q,S053G-N061P-G102A-P129S-P210S-Y217Q, S053G-N061P-G102A-P129S-Y217Q,S053G-N061P-N062Q-G097A-G100N-S101N-Y217Q,S053G-N061P-N062Q-G097A-S101N-I111V-Y217Q,S053G-N061P-S101N-G102A-P129S-Y217Q,S053G-S078N-G097A-I111V-G128S-Y217Q, S078N-G097A-G128S-Y217Q,S078N-G097A-I111V-M124V-Y217Q, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have enhanced proteolytic activity compared to BPN′ (SEQID NO:2) and a greater PI value than BPN′ in this assay. The inventionincludes a protease variant having enhanced proteolytic activitycompared to BPN′ (SEQ ID NO:2) and/or a PI value of 1.0 relative toBPN′-v3 in this BMI microswatch cleaning assay, the variant comprisingan amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 andcomprising at least one set of amino acid substitutions selected fromsaid group above, wherein amino acid positions of the variant arenumbered by correspondence with amino acid positions of the SEQ ID NO:2sequence. Also included are compositions, including, but not limited to,e.g., cleaning compositions, comprising at least one such variant andmethods for cleaning utilizing at least one such variant as described ingreater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value of about0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in DetergentComposition 4 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2)comprising at least one set of amino acid substitutions selected fromthe group consisting of N061P-G097A-M124I-Y217Q,N061P-G097A-M124V-Y217Q, N061P-N062Q-G097A-G100D-Y217Q,N061P-N062Q-G097A-G100Q-S101N-Y217Q, N061P-N062Q-G097A-G100Q-Y217Q,N061P-N062Q-G100N-G102A-Y217Q,N061P-N062Q-S078N-G097A-G100N-I111V-Y217Q,S024G-N025G-S053G-N061P-G097A-S101N-M124I-Y217Q,S053G-N061P-G097A-S101N-M124I-Y217Q,S053G-N061P-G097A-S101N-N123Q-Y217Q, wherein positions of the variantare numbered by correspondence with the sequence of SEQ ID NO:2. Suchvariants have proteolytic activity. The invention includes a proteasevariant having proteolytic activity, a PI value of 0.9 relative toBPN′-v3, and/or an enhanced proteolytic activity compared to BPN′ inthis assay, the variant comprising an amino acid sequence having atleast 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%or 99% identity to SEQ ID NO:2 and at least one set of amino acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with positionsof the SEQ ID NO:2 sequence. Also included are compositions, including,but not limited to, e.g., cleaning compositions, comprising at least onesuch variant and methods for cleaning utilizing at least one suchvariant as described in greater detail elsewhere herein.

Also provided is a subtilisin protease variant having enhancedproteolytic activity compared to BPN′ and/or a PI value of greater than1.0 compared to BPN′-v3 in a BMI microswatch cleaning assay in DetergentComposition 1 or 4 at pH 8 and 16° C., the variant comprising an aminoacid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises atleast one substitution selected from the group of X024G, X025G, X053G,X061P, X062Q, X078N, X097A, X100D/N/Q, X101N, X102A, X111V, X123A/Q/V,X124I/V, X128A/S, X129S, X210S, X217Q, and optionally at least onesubstitution selected from the group of S024G, N025G, S053G, N061P,N062Q, S078N, G097A, G100D/N/Q, S101N, G102A, I111V, N123A/Q/V, M124I/V,G128A/S, P129S, P210S, and Y217Q, wherein amino acid positions of thevariant are numbered by correspondence with positions of the sequence ofSEQ ID NO:2. Such variants have enhanced proteolytic activity comparedto BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay.Also included are compositions, including, but not limited to, e.g.,cleaning compositions, comprising at least one such variant and methodsfor cleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal toor greater than 0.5 and less than 0.9 relative to BPN′-v3 in a BMImicroswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one setof amino acid substitutions selected from the group consisting ofG097A-N123A-Y217Q, G097A-N123V-Y217Q, N061P-G102A-G128S-Y217Q,N061P-S101N-G102A-G128S-Y217Q, Y217Q, S078N-G097A-I111V-N123Q-Y217Q, andG102A-N123Q-Y217Q, wherein positions of the variant are numbered bycorrespondence with positions of the SEQ ID NO:2 sequence. Such variantshave proteolytic activity. The invention includes a protease varianthaving proteolytic activity, the variant comprising an amino acidsequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least oneset of amino acid substitutions selected from said group above, whereinpositions of the variant are numbered by correspondence with positionsof the SEQ ID NO:2 sequence. Also included are compositions, including,but not limited to, e.g., cleaning compositions, comprising at least onesuch variant and methods for cleaning utilizing at least one suchvariant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value greaterthan 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, atleast 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, atleast 2, from greater than 1.0 to about 10, from greater than 1.0 toabout 8, or from greater than 1.0 to about 5 relative to BPN′-v3 in aBMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least oneset of amino acid substitutions selected from the group consisting ofN061P-G097A-G128S-Y217Q, N061P-G097A-S101N-G128A-P201S-Y217Q,N061P-N062Q-G097A-S101N-I111V-Y217Q,S024G-N025G-N061P-G097A-S101N-G128A-P210S-Y217Q,S024G-N025G-S053G-N061P-G097A-S101N-G128A-P210S-Y217Q,S024G-N025G-S053G-N061P-G097A-S101N-G128S-Y217Q, andS024G-N025G-S053G-N061P-S078N-G097A-S101N-I111V-G128S-Y217Q, whereinamino acid positions of the variant are numbered by correspondence withthe sequence of SEQ ID NO:2. Such variants have enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′in this assay. The invention includes a protease variant having enhancedproteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value ofgreater than 1.0 to about 5 relative to BPN′-v3 in this BMI microswatchcleaning assay, the variant comprising an amino acid sequence having atleast 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%or 99% identity to SEQ ID NO:2 and comprising at least one set of aminoacid substitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal toabout 1.0 relative to BPN′-v3 in a BMI microswatch cleaning assay inDetergent Composition 1 at pH 8 and 16° C.: BPN′ amino acid sequence(SEQ ID NO:2) comprising at least one set of amino acid substitutionsselected from the group consisting of G097A-G128A-Y217Q,G097A-G128A-P210S-Y217Q, G097A-G128S-P201S-Y217Q,G097A-I111V-M124I-Y217Q, G097A-I111V-M124V-P210S-Y217Q,G097A-N123Q-P210S-Y217Q, G097A-N123Q-Y217Q,N061P-G097A-G128A-P210S-Y217Q, N061P-G097A-I111V-M124V-Y217Q,N061P-G097A-M124V-Y217Q, N061P-G097A-N123Q-Y217Q,N061P-G097A-S101N-I111V-M124V-Y217Q, N061P-G102A-P129S-Y217Q,N061P-N062Q-G097A-G100N-S101N-Y217Q, N061P-N062Q-G097A-G100Q-Y217Q,N061P-N062Q-G097A-I111V-Y217Q,N061P-N062Q-S078N-G097A-G100N-I111V-Y217Q,N061P-S078N-G097A-I111V-M124I-Y217Q,N061P-S078N-G102A-I111V-P129S-Y217Q, N062Q-G097A-I111V-P210S-Y217Q,N062Q-G097A-I111V-Y217Q, N062Q-S078N-G097A-I111V-Y217Q,S024G-N025G-S053G-N061P-G097A-S101N-I111V-M124V-Y217Q,S024G-N025G-S053G-N061P-G097A-S101N-N123Q-Y217Q,S024G-N025G-S053G-N061P-N062Q-G097A-G100N-S101N-Y217Q,S024G-N025G-S053G-N061P-N062Q-G097A-S101N-I111V-Y217Q,S024G-N025G-S053G-N061P-S101N-G102A-P129S-Y217Q,S053G-N061P-G097A-G128S-Y217Q, S053G-N061P-G102A-P129S-P210S-Y217Q,S053G-N061P-G102A-P129S-Y217Q,S053G-N061P-N062Q-G097A-S101N-I111V-Y217Q,S053G-N061P-S101N-G102A-P129S-Y217Q,S053G-S078N-G097A-I111V-G128S-Y217Q, S078N-G097A-G128S-Y217Q, andS078N-G097A-I111V-M124V-Y217Q, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have enhanced proteolytic activity compared to BPN′ (SEQID NO:2) and a greater PI value than BPN′ in this assay. The inventionincludes a protease variant having enhanced proteolytic activitycompared to BPN′ (SEQ ID NO:2) and/or a PI value of 1.0 relative toBPN′-v3 in this BMI microswatch cleaning assay, the variant comprisingan amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 andcomprising at least one set of amino acid substitutions selected fromsaid group above, wherein amino acid positions of the variant arenumbered by correspondence with amino acid positions of the SEQ ID NO:2sequence. Also included are compositions, including, but not limited to,e.g., cleaning compositions, comprising at least one such variant andmethods for cleaning utilizing at least one such variant as described ingreater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value of about0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in DetergentComposition 1 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2)comprising at least one set of amino acid substitutions selected fromthe group consisting of N061P-G097A-M124I-Y217Q,N061P-G097A-S101N-N123Q-Y217Q, N061P-N062Q-G097A-G100N-Y217Q,N061P-N062Q-G097A-G100Q-P210S-Y217Q,N061P-N062Q-G097A-G100Q-S101N-Y217Q, N061P-N062Q-G100N-G102A-Y217Q,S024G-N025G-S053G-N061P-G097A-S101N-M124I-Y217Q,S053G-N061P-G097A-M124I-Y217Q,S053G-N061P-G097A-S101N-I111V-M124V-Y217Q,S053G-N061P-G097A-S101N-M124I-Y217Q,S053G-N061P-G097A-S101N-N123Q-Y217Q, andS053G-N061P-N062Q-G097A-G100N-S101N-Y217Q, wherein positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have proteolytic activity. The invention includes aprotease variant having proteolytic activity, a PI value of 0.9 relativeto BPN′-v3, and/or an enhanced proteolytic activity compared to BPN′ inthis assay, the variant comprising an amino acid sequence having atleast 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%or 99% identity to SEQ ID NO:2 and at least one set of amino acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with positionsof the SEQ ID NO:2 sequence. Also included are compositions, including,but not limited to, e.g., cleaning compositions, comprising at least onesuch variant and methods for cleaning utilizing at least one suchvariant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal toor greater than 0.5 and less than 0.9 relative to BPN′-v3 in a BMImicroswatch cleaning assay in Detergent Composition 1 at pH 8 and 16°C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one setof amino acid substitutions selected from the group consisting ofG097A-N123A-Y217Q, G097A-N123V-Y217Q, N061P-N062Q-G097A-G100D-Y217Q,N061P-S101N-G102A-G128S-Y217Q, Y217Q, N061P-G102A-G128S-Y217Q,S078N-G097A-I111V-N123Q-Y217Q, and G102A-N123Q-Y217Q, wherein positionsof the variant are numbered by correspondence with positions of the SEQID NO:2 sequence. Such variants have proteolytic activity. The inventionincludes a protease variant having proteolytic activity, the variantcomprising an amino acid sequence having at least 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ IDNO:2 and at least one set of amino acid substitutions selected from saidgroup above, wherein positions of the variant are numbered bycorrespondence with positions of the SEQ ID NO:2 sequence. Also includedare compositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′ variants were determined to have a PI value greaterthan 1.0 to about 5 relative to BPN′-v3 in an egg microswatch cleaningassay in Detergent Composition 4 at 16° C. and pH 8: BPN′ amino acidsequence (SEQ ID NO:2) comprising the set of amino acid substitutionsN061P-G097A-S101N-G128A-P210S-Y217Q, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have enhanced proteolytic activity compared to BPN′ (SEQID NO:2) and a greater PI value than BPN′ in this assay. The inventionincludes a protease variant having enhanced proteolytic activitycompared to BPN′ (SEQ ID NO:2) and/or a PI value of greater than 1.0 toabout 5 relative to BPN′-v3 in this egg microswatch cleaning assay, thevariant comprising an amino acid sequence having at least 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity toSEQ ID NO:2 and comprising amino acid substitutionsN061P-G097A-S101N-G128A-P201S-Y217Q, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Also included are compositions, including, but not limited to, e.g.,cleaning compositions, comprising at least one such variant and methodsfor cleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal toabout 1.0 relative to BPN′-v3 in an egg microswatch cleaning assay inDetergent Composition 4 at pH 8 and 16° C.: BPN′ amino acid sequence(SEQ ID NO:2) comprising at least one set of amino acid substitutionsselected from the group consisting of G097A-G128A-Y217Q,N061P-G102A-P129S-Y217Q, N062Q-G097A-I111V-P201S-Y217Q,S024G-N025G-S053G-N061P-G097A-S101N-G128A-P210S-Y217Q,S024G-N025G-N061P-G097A-S101N-G128A-P210S-Y217Q,N061P-G097A-G128A-P201S-Y217Q, G097A-G128S-P201S-Y217Q,S024G-N025G-S053G-N061P-S078N-G097A-S101N-I111V-G128S-Y217Q,S024G-N025G-S053G-N061P-G097A-S101N-G128S-Y217Q,N061P-G097A-G128S-Y217Q, G097A-G128A-P210S-Y217Q, wherein amino acidpositions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. Such variants have enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′in this assay. The invention includes a protease variant having enhancedproteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of1.0 relative to BPN′-v3 in this egg microswatch cleaning assay, thevariant comprising an amino acid sequence having at least 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity toSEQ ID NO:2 and comprising at least one set of amino acid substitutionsselected from said group above, wherein amino acid positions of thevariant are numbered by correspondence with amino acid positions of theSEQ ID NO:2 sequence. Also included are compositions, including, but notlimited to, e.g., cleaning compositions, comprising at least one suchvariant and methods for cleaning utilizing at least one such variant asdescribed in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal toor greater than 0.5 and equal to or less than 0.9 relative to BPN′-v3 inan egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least oneset of amino acid substitutions selected from the group consisting ofN061P-G097A-M124I-Y217Q, S053G-N061P-G097A-S101N-N123Q-Y217Q,S053G-N061P-G102A-P129S-P201S-Y217Q, G097A-I111V-M124V-P201S-Y217Q,G097A-N123Q-P210S-Y217Q, S053G-N061P-S101N-G102A-P129S-Y217Q,S053G-N061P-N062Q-G097A-S101N-I111V-Y217Q,N061P-N062Q-G097A-S101N-I111V-Y217Q, N061P-N062Q-G097A-I111V-Y217Q,N062Q-G097A-I111V-Y217Q, N061P-G097A-S101N-I111V-M124V-Y217Q,G097A-N123Q-Y217Q, N061P-G097A-I111V-M124V-Y217Q,S078N-G097A-I111V-M124V-Y217Q, S053G-S078N-G097A-I111V-G128S-Y217Q,S078N-G097A-G128S-Y217Q, S053G-N061P-G097A-G128S-Y217Q,N061P-N062Q-G097A-G100N-Y217Q,S024G-N025G-S053G-N061P-G097A-S101N-N123Q-Y217Q,N061P-G097A-S101N-N123Q-Y217Q, N061P-N062Q-G097A-G100Q-P210S-Y217Q,N061P-G097A-N123Q-Y217Q,S024G-N025G-S053G-N061P-G097A-S101N-M124I-Y217Q,S053G-N061P-G097A-S101N-M124I-Y217Q, S053G-N061P-G097A-M124I-Y217Q,N061P-S078N-G097A-I111V-M124I-Y217Q, N061P-G097A-M124V-Y217Q,S024G-N025G-S053G-N061P-N062Q-G097A-G100N-S101N-Y217Q,S024G-N025G-S053G-N061P-S101N-G102A-P129S-Y217Q,N061P-S078N-G102A-I111V-P129S-Y217Q, S053G-N061P-G102A-P129S-Y217Q,S024G-N025G-S053G-N061P-N062Q-G097A-S101N-I111V-Y217Q,N062Q-S078N-G097A-I111V-Y217Q,S024G-N025G-S053G-N061P-G097A-S101N-I111V-M124V-Y217Q,S053G-N061P-G097A-S101N-I111V-M124V-Y217Q, G097A-I111V-M124I-Y217Q,Y217Q, N061P-N062Q-G100N-G102A-Y217Q,S053G-N061P-N062Q-G097A-G100N-S101N-Y217Q,N061P-N062Q-G097A-G100N-S101N-Y217Q,N061P-N062Q-S078N-G097A-G100N-I111V-Y217Q,N061P-N062Q-G097A-G100Q-Y217Q, N061P-S101N-G102A-G128S-Y217Q,G097A-N123V-Y217Q, G097A-N123A-Y217Q, G102A-N123Q-Y217Q,N061P-N062Q-G097A-G100Q-S101N-Y217Q, S078N-G097A-I111V-N123Q-Y217Q,N061P-N062Q-G097A-G100D-Y217Q, and N061P-G102A-G128S-Y217Q, whereinpositions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. Such variants have proteolytic activity. Theinvention includes a protease variant having proteolytic activity and/ora PI value of equal to or greater than 0.5 and equal to or less than 0.9relative to BPN′-v3 in this egg microswatch cleaning assay, the variantcomprising an amino acid sequence having at least 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ IDNO:2 and at least one set of amino acid substitutions selected from saidgroup above, wherein amino acid positions of the variant are numbered bycorrespondence with positions of the SEQ ID NO:2 sequence. Also includedare compositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

Also provided is a subtilisin protease variant having enhancedproteolytic activity compared to BPN′ and/or a PI value of greater than1.0 and to about 5 compared to BPN′-v3 in this egg microswatch cleaningassay in Detergent Composition 4 at pH 8 and 16° C., the variantcomprising an amino acid sequence having at least 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein thevariant comprises at least one substitution selected from the group ofX024G, X025G, X053G, X061P, X062Q, X078N, X097A, X100D/N/Q, X101N,X102A, X111V, X123A/Q/V, X124I/V, X128A/S, X129S, X210S, and X217Q, andoptionally at least one substitution selected from the group of S024G,N025G, S053G, N061P, N062Q, S078N, G097A, G100D/N/Q, S101N, G102A,I111V, N123A/Q/V, M124I/V, G128A/S, P129S, P210S, and Y217Q, whereinamino acid positions of the variant are numbered by correspondence withpositions of the sequence of SEQ ID NO:2. Such variants have enhancedproteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PIvalue than BPN′ in this assay. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

Example 6

Construction and Cleaning Performance of BPN′ Variants

A BPN′ combinatorial library based on BPN′ parent was made by DNA2.0 anddelivered as a ligation reaction. For efficient transformation of B.subtilis, DNA from the ligation reaction mixtures was amplified beforetransformation and transformants grown as described in Example 2. Thesevariants were tested for cleaning performance using BMI microswatchassay of Test Method 3 in Detergent Composition 1 and DetergentComposition 4 at 16° C. and pH 8 as well as egg microswatch assay inDetergent Composition 4 at 16° C. and pH 8. Protein content wasdetermined using the TCA assay and protease activity was assayed usingthe AAPF assay. The assays were performed as described in Example 1 andthe Performance Indices were calculated relative to BPN′-v3 (with a PIvalue of 1.0).

The following BPN′ variants were determined to have a PI value greaterthan 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, atleast 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, atleast 2, from greater than 1.0 to about 10, from greater than 1.0 toabout 8, or from greater than 1.0 to about 5 relative to BPN′-v3 in aBMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least oneset of amino acid substitutions selected from the group consisting ofS024G-N025G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,N025G-G097A-S101N-G128A-Y217Q,N025G-S038G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,N025G-S053G-N061P-S078N-G128A-Y217Q,N025G-S053G-N061P-S078N-S101N-G128A-Y217Q,N025G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,N025G-S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,N025G-S078N-G097A-S101N-G128A-Y217Q,N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,N025G-T055P-N061P-S078N-S101N-G128A-Y217Q, N061P-S101N-G128A-Y217Q,S024G-N025G-N061P-G097A-G128A-Y217Q,S024G-N025G-N061P-G097A-S101N-G128A-Y217Q,S024G-N025G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-N025G-S053G-N061P-S078N-S101N-G128A-Y217Q,S024G-N025G-S053G-T055P-G097A-S101N-G128A-Y217Q,S024G-N025G-S053G-T055P-N061P-G128A-Y217Q,S024G-N025G-T055P-G097A-G128A-Y217Q,S024G-N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-S053G-N061P-G097A-G128A-Y217Q,S024G-S053G-N061P-S078N-G097A-G128A-Y217Q,S024G-S053G-T055P-G097A-S101N-G128A-Y217Q,S024G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,S024G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-S053G-T055P-N061P-S101N-G128A-Y217Q,S024G-T055P-N061P-G097A-G128A-Y217Q, S053G-G097A-S101N-G128A-Y217Q,S053G-N061P-G097A-S101N-G128A-Y217Q-S249N,S053G-N061P-S078N-G097A-G128A-Y217Q,S053G-S078N-G097A-S101N-G128A-Y217Q,

S053G-T055P-G097A-S101N-G128A-Y217Q,S053G-T055P-N061P-S101N-G128A-Y217Q,S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,T055P-G097A-S101N-G128A-Y217Q, andT055P-N061P-S078N-G097A-S101N-G128A-Y217Q, wherein amino acid positionsof the variant are numbered by correspondence with the sequence of SEQID NO:2. Such variants have enhanced proteolytic activity compared toBPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. Theinvention includes a protease variant having enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2) and/or a PI value of greaterthan 1.0 to about 5 relative to BPN′-v3 in this BMI microswatch cleaningassay, the variant comprising an amino acid sequence having at least60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or99% identity to SEQ ID NO:2 and comprising at least one set of aminoacid substitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence.

The following BPN′ variants were determined to have a PI value equal toabout 1.0 relative to BPN′-v3 in a BMI microswatch cleaning assay inDetergent Composition 1 at pH 8 and 16° C.: BPN′ amino acid sequence(SEQ ID NO:2) comprising at least one set of amino acid substitutionsselected from the group consisting of G097A-G128S-Y217Q,G097A-G128A-Y217Q, N025G-S078N-G097A-G128A-Y217Q,N025G-T055P-G097A-G128A-Y217Q, S024G-G097A-S101N-G128A-Y217Q,S024G-I035V-T055P-N061P-S078N-G097A-Y217Q,S024G-N025G-N061P-S078N-G097A-S101N-G128A,S024G-N025G-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-N025G-N061P-S078N-S101N-G128A-Y217Q,S024G-N025G-S053G-N061P-G097A-G128A-S130G-Y217Q,S024G-N025G-S053G-N061P-G128A-Y217Q,S024G-N025G-S053G-T055P-G097A-G128A-Y217Q,S024G-N025G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q,S024G-N025G-S053G-T055P-N061P-S078N-G128A-Y217Q,S024G-N025G-S053G-T055P-S078N-S101N-G128A-Y217Q,S024G-N025G-S053G-T055P-S101N-G128A-Y217Q,S024G-N025G-T055P-G097A-S101N-G128A-Y217Q,S024G-N025G-T055P-N061P-S078N-G097A-Y217Q,S024G-N061P-G097A-S101N-G128A-Y217Q,S024G-S038G-S053G-S078N-S101N-G128A-Y217Q,S024G-S053G-S078N-G097A-S101N-G128A-Y217Q,S024G-S053G-S078N-S101N-G128A-Y217Q,S024G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q,S024G-T055P-G097A-G128A-Y217Q, S024G-T055P-N061P-G097A-S101N-G128A,S024G-T055P-N061P-S078N-S101N-G128A-Y217Q,S024G-T055P-S078N-G097A-S101N-G128A-Y217Q, S101N-G128A-Y217Q,T055P-N061P-G097A-A116S-G128A, and T055P-N061P-S078N-G128A-Y217Q,wherein amino acid positions of the variant are numbered bycorrespondence with the sequence of SEQ ID NO:2. Such variants haveenhanced proteolytic activity compared to BPN′ and a greater PI valuethan BPN′ in this assay. The invention includes a protease varianthaving enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2)and/or a PI value of 1.0 relative to BPN′-v3 in this BMI microswatchcleaning assay, the variant comprising an amino acid sequence having atleast 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%or 99% identity to SEQ ID NO:2 and comprising at least one set of aminoacid substitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

Also provided is a subtilisin protease variant having enhancedproteolytic activity compared to BPN′ and/or a PI value of greater than1.0 compared to BPN′-v3 in this BMI microswatch cleaning assay inDetergent Composition 1 at pH 8 and 16° C., the variant comprising anamino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprisesat least one substitution selected from the group of X024G, X025G,X035V, X038G, X053G, X055P, X061P, X078N, X097A, X101N, X116S, X128A/S,X130G, X216Q, X217Q, and X249N, and optionally at least one substitutionselected from the group of S024G, N025G, I035V, S038G, S053G, T055P,N061P, S078N, G097A, S101N, A116S, G128A/S, S130G, Y216Q, Y217Q, andS249N, wherein amino acid positions of the variant are numbered bycorrespondence with positions of the sequence of SEQ ID NO:2. Alsoincluded are compositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′ variants were determined to have a PI value greaterthan 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, atleast 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, atleast 2, from greater than 1.0 to about 10, from greater than 1.0 toabout 8, or from greater than 1.0 to about 5 relative to BPN′-v3 in aBMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least oneset of amino acid substitutions selected from the group consisting ofS024G-N025G-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-N025G-S053G-N061P-S078N-S101N-G128A-Y217Q,S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,S024G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,S024G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-T055P-N061P-S078N-S101N-G128A-Y217Q,S053G-G097A-S101N-G128A-Y217Q, and T055P-N061P-S078N-G128A-Y217Q,wherein amino acid positions of the variant are numbered bycorrespondence with the sequence of SEQ ID NO:2. Such variants haveenhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and agreater PI value than BPN′ in this assay. The invention includes aprotease variant having enhanced proteolytic activity compared to BPN′(SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relativeto BPN′-v3 in this BMI microswatch cleaning assay, the variantcomprising an amino acid sequence having at least 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ IDNO:2 and comprising at least one set of amino acid substitutionsselected from said group above, wherein amino acid positions of thevariant are numbered by correspondence with amino acid positions of theSEQ ID NO:2 sequence.

The following BPN′ variants were determined to have a PI value equal toabout 1.0 relative to BPN′-v3 in a BMI microswatch cleaning assay ofTest Method 3 in Detergent Composition 4 at pH 8 and 16° C.: BPN′ aminoacid sequence (SEQ ID NO:2) comprising at least one set of amino acidsubstitutions selected from the group consisting of G097A-G128S-Y217Q,G097A-G128A-Y217Q, N025G-G097A-S101N-G128A-Y217Q,N025G-S038G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,N025G-S053G-N061P-S078N-G128A-Y217Q,N025G-S053G-N061P-S078N-S101N-G128A-Y217Q,N025G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,N025G-S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,N025G-S078N-G097A-G128A-Y217Q, N025G-S078N-G097A-S101N-G128A-Y217Q,N025G-T055P-G097A-G128A-Y217Q,N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,N025G-T055P-N061P-S078N-S101N-G128A-Y217Q, N061P-S101N-G128A-Y217Q,S024G-G097A-S101N-G128A-Y217Q,S024G-I035V-T055P-N061P-S078N-G097A-Y217Q,S024G-N025G-N061P-G097A-G128A-Y217Q,S024G-N025G-N061P-G097A-S101N-G128A-Y217Q,S024G-N025G-N061P-S078N-G097A-S101N-G128A,S024G-N025G-N061P-S078N-S101N-G128A-Y217Q,S024G-N025G-S053G-N061P-G097A-G128A-S130G-Y217Q,S024G-N025G-S053G-N061P-G128A-Y217Q,S024G-N025G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-N025G-S053G-T055P-G097A-G128A-Y217Q,S024G-N025G-S053G-T055P-G097A-S101N-G128A-Y217Q,S024G-N025G-S053G-T055P-N061P-G128A-Y217Q,S024G-N025G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q,S024G-N025G-S053G-T055P-N061P-S078N-G128A-Y217Q,S024G-N025G-S053G-T055P-S078N-S101N-G128A-Y217Q,S024G-N025G-S053G-T055P-S101N-G128A-Y217Q,S024G-N025G-T055P-G097A-G128A-Y217Q,S024G-N025G-T055P-G097A-S101N-G128A-Y217Q,S024G-N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-N061P-G097A-S101N-G128A-Y217Q,S024G-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-S038G-S053G-S078N-S101N-G128A-Y217Q,S024G-S053G-N061P-G097A-G128A-Y217Q,S024G-S053G-N061P-S078N-G097A-G128A-Y217Q,S024G-S053G-S078N-G097A-S101N-G128A-Y217Q,S024G-S053G-S078N-S101N-G128A-Y217Q,S024G-S053G-T055P-G097A-S101N-G128A-Y217Q,S024G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q,S024G-S053G-T055P-N061P-S101N-G128A-Y217Q,S024G-T055P-G097A-G128A-Y217Q, S024G-T055P-N061P-G097A-G128A-Y217Q,S024G-T055P-N061P-G097A-S101N-G128A,S024G-T055P-S078N-G097A-S101N-G128A-Y217Q,S053G-N061P-G097A-S101N-G128A-Y217Q-S249N,S053G-N061P-S078N-G097A-G128A-Y217Q,S053G-S078N-G097A-S101N-G128A-Y217Q,S053G-T055P-G097A-S101N-G128A-Y217Q,S053G-T055P-N061P-S101N-G128A-Y217Q,S053G-T055P-S078N-G097A-S101N-G128A-Y217Q, S101N-G128A-Y217Q,T055P-G097A-S101N-G128A-Y217Q, andT055P-N061P-S078N-G097A-S101N-G128A-Y217Q, wherein amino acid positionsof the variant are numbered by correspondence with the sequence of SEQID NO:2. Such variants have enhanced proteolytic activity compared toBPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. Theinvention includes a protease variant having enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2) and/or a PI value of 1.0relative to BPN′-v3 in this BMI microswatch cleaning assay, the variantcomprising an amino acid sequence having at least 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ IDNO:2 and comprising at least one set of amino acid substitutionsselected from said group above, wherein amino acid positions of thevariant are numbered by correspondence with amino acid positions of theSEQ ID NO:2 sequence. Also included are compositions, including, but notlimited to, e.g., cleaning compositions, comprising at least one suchvariant and methods for cleaning utilizing at least one such variant asdescribed in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value of about0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay of TetsMethod 3 in Detergent Composition 4 at pH 8 and 16° C.: BPN′ amino acidsequence (SEQ ID NO:2) comprising at least one set of amino acidsubstitutions selected from the group consisting ofT055P-N061P-G097A-A116S-G128A,S024G-N025G-T055P-N061P-S078N-G097A-Y217Q, wherein positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have proteolytic activity and may have enhancedproteolytic activity compared to BPN′ (SEQ ID NO:2) in this assay. Theinvention includes a protease variant having proteolytic activity, a PIvalue of 0.9 relative to BPN′-v3, and/or an enhanced proteolyticactivity compared to BPN′ in this assay, the variant comprising an aminoacid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at leastone set of amino acid substitutions selected from said group above,wherein amino acid positions of the variant are numbered bycorrespondence with positions of the SEQ ID NO:2 sequence. Also includedare compositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′ variants were determined to have a PI value greaterthan 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, atleast 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, atleast 2, from greater than 1.0 to about 10, from greater than 1.0 toabout 8, or from greater than 1.0 to about 5 relative to BPN′-v3 in anegg microswatch cleaning assay in Detergent Composition 4 at pH 8 and16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least oneset of amino acid substitutions selected from the group consisting ofN025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,N061P-S101N-G128A-Y217Q,S024G-N025G-S053G-N061P-S078N-S101N-G128A-Y217Q,S024G-N025G-S053G-T055P-S101N-G128A-Y217Q,S024G-N025G-S053G-T055P-N061P-G128A-Y217Q,S024G-N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,N025G-S053G-N061P-S078N-G128A-Y217Q,S024G-N025G-S053G-T055P-G097A-S101N-G128A-Y217Q,S024G-N025G-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-S053G-T055P-G097A-S101N-G128A-Y217Q,S053G-N061P-S078N-G097A-G128A-Y217Q,S024G-N025G-T055P-G097A-G128A-Y217Q,S024G-N025G-S053G-T055P-G097A-G128A-Y217Q, wherein amino acid positionsof the variant are numbered by correspondence with the sequence of SEQID NO:2. Such variants have enhanced proteolytic activity compared toBPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. Theinvention includes a protease variant having enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2) and/or a PI value of greaterthan 1.0 to about 5 relative to BPN′-v3 in this egg microswatch cleaningassay, the variant comprising an amino acid sequence having at least60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or99% identity to SEQ ID NO:2 and comprising at least one set of aminoacid substitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence.

The following BPN′ variants were determined to have a PI value equal toabout 1.0 relative to BPN′-v3 in an egg microswatch cleaning assay inDetergent Composition 4 at pH 8 and 16° C.: BPN′ amino acid sequence(SEQ ID NO:2) comprising at least one set of amino acid substitutionsselected from the group consisting of G097A-G128S-Y217Q,G097A-G128A-Y217Q, S024G-G097A-S101N-G128A-Y217Q,N025G-T055P-N061P-S078N-S101N-G128A-Y217Q,S053G-T055P-N061P-S101N-G128A-Y217Q,S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,N025G-S053G-N061P-S078N-S101N-G128A-Y217Q,S024G-S053G-T055P-N061P-S101N-G128A-Y217Q,S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,N025G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-N025G-N061P-S078N-S101N-G128A-Y217Q,S024G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,T055P-N061P-S078N-G128A-Y217Q,N025G-S038G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-N025G-S053G-N061P-G128A-Y217Q,S024G-N025G-S053G-T055P-S078N-S101N-G128A-Y217Q,T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-N025G-S053G-T055P-N061P-S078N-G128A-Y217Q,S024G-N025G-N061P-G097A-S101N-G128A-Y217Q,S024G-T055P-G097A-G128A-Y217Q, T055P-N061P-G097A-A116S-G128A,S053G-T055P-G097A-S101N-G128A-Y217Q, T055P-G097A-S101N-G128A-Y217Q,S024G-N061P-G097A-S101N-G128A-Y217Q,S024G-N025G-N061P-G097A-G128A-Y217Q,S024G-S053G-S078N-S101N-G128A-Y217Q,S024G-N025G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q,S053G-G097A-S101N-G128A-Y217Q, N025G-T055P-G097A-G128A-Y217Q,S024G-T055P-S078N-G097A-S101N-G128A-Y217Q,N025G-S078N-G097A-S101N-G128A-Y217Q, N025G-G097A-S101N-G128A-Y217Q,S024G-S053G-N061P-S078N-G097A-G128A-Y217Q,S024G-N025G-T055P-N061P-S078N-G097A-Y217Q,S024G-I035V-T055P-N061P-S078N-G097A-Y217Q, wherein amino acid positionsof the variant are numbered by correspondence with the sequence of SEQID NO:2. Such variants have enhanced proteolytic activity compared toBPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. Theinvention includes a protease variant having enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2) and/or a PI value of 1.0relative to BPN′-v3 in this egg microswatch cleaning assay, the variantcomprising an amino acid sequence having at least 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ IDNO:2 and comprising at least one set of amino acid substitutionsselected from said group above, wherein amino acid positions of thevariant are numbered by correspondence with amino acid positions of theSEQ ID NO:2 sequence. Also included are compositions, including, but notlimited to, e.g., cleaning compositions, comprising at least one suchvariant and methods for cleaning comprising utilizing at least one suchvariant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value of about0.9 relative to BPN′-v3 in an egg microswatch cleaning assay inDetergent Composition 4 at pH 8 and 16° C.: BPN′ amino acid sequence(SEQ ID NO:2) comprising at least one set of amino acid substitutionsselected from the group consisting of S101N-G128A-Y217Q,S024G-T055P-N061P-G097A-S101N-G128A,S024G-N025G-N061P-S078N-G097A-S101N-G128A,S024G-T055P-N061P-S078N-S101N-G128A-Y217Q,S024G-N025G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,S053G-N061P-G097A-S101N-G128A-Y217Q-S249N,N025G-S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,S024G-N025G-T055P-G097A-S101N-G128A-Y217Q,S024G-S053G-N061P-G097A-G128A-Y217Q,S024G-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q,S024G-T055P-N061P-G097A-G128A-Y217Q,S024G-S038G-S053G-S078N-S101N-G128A-Y217Q,S053G-S078N-G097A-S101N-G128A-Y217Q, N025G-S078N-G097A-G128A-Y217Q, andS024G-N025G-S053G-N061P-G097A-G128A-S130G-Y217Q, wherein positions ofthe variant are numbered by correspondence with the sequence of SEQ IDNO:2. Such variants have proteolytic activity. The invention includes aprotease variant having proteolytic activity, a PI value of 0.9 relativeto BPN′-v3, and/or an enhanced proteolytic activity compared to BPN′ inthis assay, the variant comprising an amino acid sequence having atleast 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%or 99% identity to SEQ ID NO:2 and at least one set of amino acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with positionsof the SEQ ID NO:2 sequence. Also included are compositions, including,but not limited to, e.g., cleaning compositions, comprising at least onesuch variant and methods for cleaning utilizing at least one suchvariant as described in greater detail elsewhere herein.

The following BPN′ variant was determined to have a PI value of about0.8 relative to BPN′-v3 in an egg microswatch cleaning assay inDetergent Composition 4 at pH 8 and 16° C.: BPN′ amino acid sequence(SEQ ID NO:2) comprising amino acid substitutionsS024G-S053G-S078N-G097A-S101N-G128A-Y217Q, wherein positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.The invention includes a protease variant having proteolytic activity, aPI value of 0.8 relative to BPN′-v3, and/or an enhanced proteolyticactivity compared to BPN′ in this assay, the variant comprising an aminoacid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprisingamino acid substitutions S024G-S053G-S078N-G097A-S101N-G128A-Y217Q,wherein amino acid positions of the variant are numbered bycorrespondence with positions of the SEQ ID NO:2 sequence. Also includedare compositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′ variants were determined to have a PI value greaterthan 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, atleast 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, atleast 2, from greater than 1.0 to about 10, from greater than 1 to about12, from greater than 4 to about 12, from greater than 1.0 to about 8,or from greater than 1.0 to about 5 relative to BPN′-v3 in an AAPFproteolytic assay: BPN′ amino acid sequence (SEQ ID NO:2) comprising atleast one set of amino acid substitutions selected from the groupconsisting of S024G-G097A-S101N-G128A-Y217Q, S101N-G128A-Y217Q,N025G-T055P-N061P-S078N-S101N-G128A-Y217Q,S053G-T055P-N061P-S101N-G128A-Y217Q,S024G-T055P-N061P-G097A-S101N-G128A,S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,N025G-S053G-N061P-S078N-S101N-G128A-Y217Q,S024G-S053G-T055P-N061P-S101N-G128A-Y217Q,S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,S024G-N025G-N061P-S078N-G097A-S101N-G128A, N061P-S101N-G128A-Y217Q,S024G-N025G-S053G-N061P-S078N-S101N-G128A-Y217Q,S024G-T055P-N061P-S078N-S101N-G128A-Y217Q,N025G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-N025G-S053G-T055P-S101N-G128A-Y217Q,N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-N025G-N061P-S078N-S101N-G128A-Y217Q,S024G-N025G-S053G-T055P-N061P-G128A-Y217Q,S024G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,S024G-N025G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,T055P-N061P-S078N-G128A-Y217Q,S024G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,N025G-S038G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-N025G-S053G-N061P-G128A-Y217Q,N025G-S053G-N061P-S078N-G128A-Y217Q,S024G-N025G-S053G-T055P-G097A-S101N-G128A-Y217Q,S024G-N025G-S053G-T055P-S078N-S101N-G128A-Y217Q,T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-N025G-S053G-T055P-N061P-S078N-G128A-Y217Q,S024G-N025G-N061P-G097A-S101N-G128A-Y217Q,S053G-N061P-G097A-S101N-G128A-Y217Q-S249N,N025G-S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,S024G-T055P-G097A-G128A-Y217Q, T055P-N061P-G097A-A116S-G128A,S024G-N025G-T055P-G097A-S101N-G128A-Y217Q,S024G-N025G-N061P-S078N-G097A-S101N-G128A-Y217Q,S053G-T055P-G097A-S101N-G128A-Y217Q, T055P-G097A-S101N-G128A-Y217Q,S024G-N061P-G097A-S101N-G128A-Y217Q,S024G-S053G-T055P-G097A-S101N-G128A-Y217Q, G097A-G128S-Y217Q,S024G-S053G-N061P-G097A-G128A-Y217Q,S024G-N025G-N061P-G097A-G128A-Y217Q,S024G-N061P-S078N-G097A-S101N-G128A-Y217Q,S024G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q,S024G-S053G-S078N-S101N-G128A-Y217Q,S024G-N025G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q,S053G-N061P-S078N-G097A-G128A-Y217Q,S024G-T055P-N061P-G097A-G128A-Y217Q,S024G-S038G-S053G-S078N-S101N-G128A-Y217Q,S053G-G097A-S101N-G128A-Y217Q, N025G-T055P-G097A-G128A-Y217Q,S024G-T055P-S078N-G097A-S101N-G128A-Y217Q,S053G-S078N-G097A-S101N-G128A-Y217Q,S024G-N025G-T055P-G097A-G128A-Y217Q,S024G-N025G-S053G-T055P-G097A-G128A-Y217Q,N025G-S078N-G097A-S101N-G128A-Y217Q, N025G-G097A-S101N-G128A-Y217Q,S024G-S053G-N061P-S078N-G097A-G128A-Y217Q,S024G-S053G-S078N-G097A-S101N-G128A-Y217Q,N025G-S078N-G097A-G128A-Y217Q, andS024G-N025G-S053G-N061P-G097A-G128A-S130G-Y217Q, wherein amino acidpositions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. Such variants have enhanced proteolyticactivity compared to BPN′ protease (SEQ ID NO:2) and a greater PI valuethan BPN′ in this assay. The invention includes a protease varianthaving enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2)and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v3 inthis AAPF assay, the variant comprising an amino acid sequence having atleast 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%or 99% identity to SEQ ID NO:2 and comprising at least one set of aminoacid substitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence.

The following BPN′ variant was determined to have a PI value equal toabout 1.0 relative to BPN′-v3 in an AAPF proteolytic assay: BPN′ aminoacid sequence (SEQ ID NO:2) comprising amino acid substitutionsG097A-G128A-Y217Q, wherein amino acid positions of the variant arenumbered by correspondence with the sequence of SEQ ID NO:2. Suchvariants have enhanced proteolytic activity compared to BPN′ protease(SEQ ID NO:2) and a greater PI value than BPN′ in this assay. Theinvention includes a protease variant having enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2) and/or a PI value of 1.0relative to BPN′-v3 in this AAPF assay, the variant comprising an aminoacid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprisingamino acid substitutions G097A-G128A-Y217Q, wherein amino acid positionsof the variant are numbered by correspondence with amino acid positionsof the SEQ ID NO:2 sequence. Also included are compositions, including,but not limited to, e.g., cleaning compositions, comprising at least onesuch variant and methods for cleaning utilizing at least one suchvariant as described in greater detail elsewhere herein.

Example 7 Construction of Site Evaluation Libraries of BPN′-v36 andCleaning Performance of BPN′-v36 Variants

Construction of the Site Evaluation Libraries of BPN′-v36

The amino acid sequence of BPN′-v36 is that of SEQ ID NO:6 and thenucleic acid sequence encoding the BPN′-v36 protease variant is that ofSEQ ID NO:5.

The amino acid sequence of BPN′-v36 may be represented by reference tothe subtilisin BPN′ amino acid sequence of SEQ ID NO:2. That is,BPN′-v36 may be represented as the subtilisin BPN′ sequence of SEQ IDNO:2 with the six amino acid substitutionsS024G-S053G-S078N-S101N-G128A-Y217Q. The BPN′-v36 amino acid sequencemay be conveniently designated asBPN′-S024G-S053G-S078N-S101N-G128A-Y217Q orBPN′+S024G+S053G+S078N+S101N+G128A+Y217Q. Throughout this specification,unless otherwise indicated, each amino acid position of an amino acidsequence is numbered according to the numbering of a corresponding aminoacid position in the amino acid sequence of Bacillus amyloliquefacienssubtilisin BPN′ shown in SEQ ID NO:2 as determined by alignment of thevariant amino acid sequence with the Bacillus amyloliquefacienssubtilisin BPN′ amino acid sequence.

Site evaluation libraries (SELs) were created at every single amino acidposition in mature BPN′-v36 (i.e.,BPN′-S24G-S53G-S78N-S101N-G128A-Y217Q) protein by PCR fusion.

For each codon to be mutated in the BPN′-v36 protease, a pair ofpartially overlapping, complementary (mutagenic forward and reverse)primers were designed. Each mutagenic primer contained the NNS (N=A,C,G,or T and S=G or C) mutagenic codon in the center flanked by at least 15nucleotides on each side. To create a library at a given position, twoPCR reactions were carried out using either a common forwardgene-flanking primer (P4974, SEQ ID NO:60) and a mutagenic NNS reverseprimer, or the common reverse gene-flanking primer (P4976, SEQ ID NO:61)and a mutagenic NNS forward primer. These PCR reactions generated twoPCR fragments, one encoding the 5′ half of the mutant BPN′-v36 gene (5′gene fragment) and the other encoding the 3′ half of the mutant BPN′-v36gene (3′ gene fragment).

Each PCR amplification reaction contained 30 pmol of each primer and 100ng of the BPN′-v36 parent template DNA (plasmid pHPLT-BPN′-v36, see FIG.4) Amplifications were carried out using Vent DNA polymerase (NEB). ThePCR reaction (20 μL) was initially heated at 95° C. for 2.5 min followedby 30 cycles of denaturation at 94° C. for 15 sec., annealing at 55° C.for 15 sec. and extension at 72° C. for 40 sec. Following amplification,the 5′ and 3′ gene fragments were gel-purified by the QIAGEN® gel-bandpurification kit, mixed (50 ng of each fragment), mixed and amplified byPCR once again using the primers P4973 (SEQ ID NO:62) and P4950 (SEQ IDNO:63) to generate the full-length gene fragment. The PCR conditionswere same as described above, except the extension phase, which wascarried out at 72° C. for 2 min. The full-length DNA fragment wasgel-purified by the QIAGEN® gel-band purification kit, digested by theBamHI and HindIII restriction enzymes and ligated with the pHPLT-BPN′partial opt vector that also was digested with the same restrictionenzymes. Ligation mixtures were amplified using rolling circleamplification in an Illustra Templiphi kit according to themanufacturer's recommendation (GE Healthcare) to generate multimeric DNAfor transformation into Bacillus subtilis. For this purpose, 1 μl of theligation mixture was mixed with 5 μl of the sample buffer, heated to 95°C. for 3 min and cooled on ice. Next, 5 μl of the reaction buffer and0.2 μl of the enzyme were added to each tube, followed by incubation at30° C. for 10 hours. Products of the rolling circle amplification werediluted 100 times and used to transform B. subtilis cells (ΔaprE, ΔnprE,amyE::xylRPxylAcomK-phleo). An aliquot of the transformation mix wasplated on LB plates containing 1.6% skim milk and 10 μg/mL neomycin andincubated overnight at 37° C. Subsequently, the colonies with halos wereinoculated in 150 μl of LB media containing 10 μg/mL neomycin. The nextday, cultures were either frozen with 15% glycerol or grown in MBDmedium for biochemical analysis as described in Example 2.

Cleaning Performance of the BPN′-v36 Variants

Protein variants from BPN′-v36 SEL were tested for cleaning performanceusing a BMI microswatch assay in Detergent Composition 4 at 16° C. andpH 8 and egg microswatch assay in Detergent Composition 4 at 16° C. andpH 8. Protein content was determined using the TCA assay. The assayswere performed as described in Example 1 and the Performance Indiceswere calculated relative to BPN′-v36 (i.e.,BPN′-S24G-S53G-S78N-S101N-G128A-Y217Q).

The following BPN′-v36 variants were determined to have a PI valuegreater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from greater than 1.0 to about 10, from greater than1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q (SEQ ID NO:6)(i.e., BPN′-v36) comprising at least one amino acid substitutionselected from the group consisting of A116V, G160S, I111L, I115V, N109S,N117M, P005G, Q059V, T164S, Y262M, A015Q, A015S, A098E, A098N, A098S,A098T, A098V, A098Y, A114S, A114T, A116G, A116L, A116S, A116T, A116W,A133G, A133H, A133T, A133V, A137G, A137I, A137L, A137S, A137T, A138S,A216E, A216F, A216V, D099S, D181E, F261A, F261Q, G024F, G024I, G024Q,G024Y, G097S, G160T, G211L, G211V, H017F, H017W, H039V, H226A, I031V,I111V, I268V, K170R, K265R, L016Q, L016T, L135M, L209T, L209V, L233M,L257T, L257V, L267A, L267V, N025A, N025I, N025Q, N025R, N025T, N025V,N101I, N101Q, N101S, N109A, N109G, N109H, N109L, N109M, N109Q, N109T,N117Q, N184A, N184L, N184T, N184W, N212G, N212L, N212V, N243P, N252G,N252M, P005T, P014S, P040G, P040L, P040Q, P129A, P129S, P172G, P172S,P194Q, P210A, P210S, Q185F, Q185G, Q185I, Q185M, Q185N, Q185S, Q275H,R186K, S009A, S009G, S009H, S009M, S018T, S130T, S132N, S145K, S159T,S161I, S161K, S161N, S161T, S162I, S162M, S162Y, S163G, S182F, S182G,S182V, S182W, S183F, S183L, S183M, S183T, S183V, S183W, S224A, S236T,S249V, T022A, T022G, T022Q, T022V, T208V, T242S, T253N, T253S, T254A,T254S, T255L, T255S, T255V, V004A, V004P, V004W, V084C, V139C, V165M,V203F, Y021K, Y021N, Y021T, Y021V, Y167F, Y171F, Y214F, Y262F, andY262T, wherein amino acid positions of the variant are numbered bycorrespondence with the sequence of SEQ ID NO:2. Such variants haveenhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and agreater PI value than BPN′ in this assay. The invention includes aprotease variant having enhanced proteolytic activity compared to BPN′(SEQ ID NO:2), enhanced proteolytic activity compared to BPN′-v3 andBPN′-v36, a PI value greater than that of BPN′-v3, and/or a PI valuegreater than 1 to about 5 relative to BPN′-v36 in this BMI microswatchcleaning assay, the variant comprising an amino acid sequence having atleast 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at leastone, two, three, four, five, six or more amino acid substitutionsselected from said group above, wherein amino acid positions of thevariant are numbered by correspondence with amino acid positions of theSEQ ID NO:2 sequence. Also included are compositions, including, but notlimited to, e.g., cleaning compositions, comprising at least one suchvariant and methods for cleaning utilizing at least one such variant asdescribed in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto about 1.0 relative to BPN′-v36 in a BMI microswatch cleaning assay inDetergent Composition 4 at pH 8 and 16° C.:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one amino acid substitution selected from thegroup consisting of BPN′-v36, A001G, A001Y, A013G, A013V, A015F, A015G,A015K, A015M, A015P, A015T, A015W, A015Y, A029G, A073S, A088C, A088I,A088L, A088T, A088V, A098D, A098K, A098P, A098R, A098W, A116D, A116E,A116R, A128S, A133L, A133M, A133S, A134G, A134S, A137N, A137V, A144M,A144Q, A144S, A144T, A144V, A151C, A176S, A176T, A179S, A216G, A216L,A216P, A216Q, A216S, A216T, A216Y, A228T, A230C, A231C, A272I, A272L,A272Q, A272S, A272T, A272W, A273S, A274G, A274L, A274Q, A274T, A274V,D041E, D099G, D099N, D120A, D120K, D120Q, D120R, D120S, D140E, D181S,D259E, E054D, E156D, E156T, E251V, F261G, F261H, F261L, F261R, F261S,F261T, F261V, F261W, G007A, G007S, G020A, G020D, G020S, G024N, G024R,G024S, G024T, G024V, G024W, G053H, G053K, G053N, G053T, G097A, G097D,G097T, G131A, G131H, G131P, G131Q, G131T, G131V, G160H, G160P, G166A,G166S, G166T, G211A, G211D, G211M, G211N, G211P, G211Q, G211R, G211W,G215A, G215N, G215V, H017L, H017M, H017T, H017V, H017Y, H039A, H039C,H039N, H226F, H226I, H226M, H226S, H226V, H226Y, I035V, I079A, I079S,I079T, I079V, I079W, I108V, I115L, I122A, I234L, I234V, K012R, K027R,K136R, K141F, K213W, K237R, K256R, K265Q, L016A, L016F, L016I, L016S,L016V, L042I, L075A, L075M, L075Q, L075V, L075Y, L082K, L082M, L082Q,L082V, L090I, L196I, L196V, L209Q, L209W, L233A, L233Q, L233V, L235I,L235K, L250I, L257A, L257H, L257Q, L257S, L257Y, L267Q, L267R, L267S,L267T, M199V, N025F, N025G, N025H, N025K, N025L, N025M, N025S, N025Y,N061F, N061H, N061P, N061S, N061T, N061V, N061W, N076G, N076W, N078S,N078T, N078V, N101A, N101H, N101L, N101T, N109K, N109R, N117A, N117E,N117H, N117K, N118G, N184G, N184H, N184I, N184S, N184V, N212A, N212F,N212I, N212K, N212P, N212Q, N212S, N212Y, N218A, N218H, N218L, N218S,N240E, N240H, N240L, N240R, N240T, N243A, N243Q, N243T, N243V, N252A,N252K, N252L, N252Q, N252R, N252S, N252T, N269Q, N269S, P014G, P014Q,P014T, P040A, P040H, P040S, P040T, P040V, P040Y, P086A, P086C, P086F,P086H, P086S, P129D, P129G, P129K, P129T, P172A, P172Q, P194A, P194G,P194L, P194M, P194S, P194V, P194Y, P210G, P210R, P210V, Q002A, Q002S,Q010A, Q010F, Q010H, Q010I, Q010L, Q010N, Q010S, Q010T, Q019A, Q019G,Q019N, Q019S, Q019T, Q019V, Q019W, Q059I, Q103L, Q103S, Q185A, Q185H,Q185L, Q185T, Q185Y, Q206P, Q206S, Q206Y, Q217I, Q217N, Q217S, Q217T,Q245K, Q275D, Q275S, Q275W, S003A, S003G, S003H, S003M, S003P, S003Q,S003T, S003V, S009I, S009L, S009P, S009T, S009W, S018A, S018G, S018I,S018L, S018M, S018N, S018P, S018V, S018W, S033T, S037Q, S037T, S037V,S038G, S038H, S038K, S038Q, S038T, S063K, S063N, S063Q, S063T, S087A,S087F, S087G, S087Q, S087T, S089L, S089M, S089N, S089Q, S089T, S089W,S130A, S130F, S130G, S130L, S130V, S145A, S145H, S145M, S145V, S159A,S159G, S159H, S159Q, S159R, S161A, S161G, S161H, S161L, S161M, S161P,S161Q, S161W, S162A, S162F, S162G, S162L, S162N, S162P, S162R, S162V,S163P, S173A, S173G, S182A, S182H, S182K, S182L, S182N, S182P, S182Q,S182T, S183A, S183G, S183H, S183Q, S188A, S188G, S188T, S188V, S191A,S204A, S204I, S204L, S204Q, S204V, S224C, S236A, S236N, S236Q, S248A,S248F, S248G, S248I, S248K, S248L, S248M, S248N, S248Q, S248T, S248V,S249A, S249C, S249H, S249Q, S249T, S249W, S249Y, S260H, S260N, S260P,S260T, T022H, T022K, T022N, T022R, T022S, T022Y, T055A, T055G, T055L,T055N, T055P, T055Q, T071S, T158H, T158S, T164N, T208C, T208L, T220S,T242N, T244A, T244G, T244H, T244I, T244Q, T244S, T244V, T244W, T253A,T253G, T253H, T253Q, T254V, T255A, T255G, T255H, T255I, T255Q, T255Y,V004G, V004N, V004R, V008A, V008C, V008M, V026I, V044I, V044L, V045H,V045K, V045L, V045M, V045Q, V045S, V045V, V045W, V045Y, V051I, V081L,V081Q, V081T, V084A, V084S, V084T, V093I, V121I, V143N, V143S, V143Y,V147C, V147I, V147L, V147T, V180I, V180L, V180T, V192A, V192S, V192T,V198I, V198L, V198M, V203H, V203I, V203L, V203N, V203Q, V203T, V203W,V203Y, V270A, V270S, V270T, W241M, W241Y, Y006G, Y006H, Y006I, Y006K,Y006L, Y006P, Y006Q, Y006T, Y006V, Y006W, Y021A, Y021D, Y021E, Y021L,Y021Q, Y021R, Y021S, Y104F, Y104I, Y214L, Y214V, Y214W, Y262A, Y262G,Y262L, Y262N, Y262S, Y262W, Y263G, and Y263W, wherein amino acidpositions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. Thus, e.g., the invention includesBPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising substitution A128S, e.g.,BPN′-S024G-S053G-S078N-S101N-G128S-Y217Q. Such variants have enhancedproteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PIvalue than BPN′ in this assay. The invention includes a protease varianthaving enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), aPI value of about 1.0 relative to BPN′-v3, and/or a PI value of 1.0relative to BPN′-v36 in this BMI microswatch cleaning assay, the variantcomprising an amino acid sequence having at least 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ IDNO:2 or SEQ ID NO:6 and comprising at least one, two, three, four, five,six or more amino acid substitutions selected from said group above,wherein amino acid positions of the variant are numbered bycorrespondence with amino acid positions of the SEQ ID NO:2 sequence.Also included are compositions, including, but not limited to, e.g.,cleaning compositions, comprising at least one such variant and methodsfor cleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′-v36 variants were determined using Test Method 3 tohave a PI value equal to about 0.9 relative to BPN′-v36 in a BMImicroswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one amino acid substitution selected from thegroup consisting of A001F, A001K, A001L, A001M, A001Q, A001R, A001S,A001T, A001V, A013C, A013S, A015D, A015E, A015L, A015R, A048S, A073N,A073T, A074G, A074S, A085C, A085G, A085S, A085V, A088M, A088S, A092S,A098G, A114G, A133P, A137E, A137H, A144G, A144H, A144K, A144L, A144N,A153S, A153V, A176C, A179G, A187G, A187S, A200G, A216W, A223S, A228S,A230T, A230V, A231V, A232C, A232V, A272E, A272G, A272K, A272P, A273G,A273L, A273V, A274M, A274R, D036E, D099A, D099Q, D120E, D181A, D181G,D259A, D259G, D259Q, D259T, E156A, E156S, E251I, E251L, E251Q, E251T,F058Y, F261C, F261D, F261K, F261P, G020E, G020F, G020H, G020L, G020N,G020Q, G020R, G020T, G020Y, G024A, G024P, G053A, G053D, G053E, G053F,G053L, G053Q, G053S, G053Y, G097K, G097M, G157A, G157S, G160A, G160L,G166C, G166I, G166Q, G169A, G211K, G215H, G215L, G215S, G215T, G215W,G258S, H017I, H039S, H226L, H238N, H238Y, I011L, I011V, I031L, I079F,I079K, I079L, I079M, I079Q, I205A, I205V, I268L, I268M, K012G, K043F,K043H, K043I, K043N, K043Q, K043T, K141A, K141R, K141W, K170A, K213A,K213G, K213H, K213I, K213L, K213N, K213Q, K213R, K213S, K213T, K213V,K237A, K237H, K237I, K237L, K237N, K237S, K256A, K256G, K256H, K256M,K256P, K256Q, K256W, K265H, L016E, L042V, L075G, L075H, L075I, L075T,L082A, L082F, L082H, L082R, L082S, L082T, L090M, L135F, L196M, L209C,L209H, L209S, L233S, L235M, L235R, L235W, L257C, L257G, L267F, M050Y,M119C, M119I, M124L, N025C, N025E, N025P, N061A, N061G, N061I, N061K,N061L, N061Q, N061R, N062S, N062T, N076A, N076P, N076Q, N076S, N076T,N076V, N078G, N078H, N078K, N078P, N078Q, N078R, N101F, N117R, N117S,N118D, N118H, N118Q, N118R, N118S, N118T, N184C, N184E, N184R, N212D,N212R, N212W, N218F, N218G, N218M, N218P, N218T, N218V, N218W, N240A,N240G, N240Q, N240S, N240W, N243C, N243G, N243S, N252V, N269H, P005A,P005D, P005M, P005Q, P014A, P014M, P014R, P014V, P040F, P040R, P040W,P129E, P129R, P172E, P172K, P194H, P194R, P194W, P201A, P201G, P210L,P239K, P239R, Q002D, Q002E, Q002G, Q002I, Q002P, Q002V, Q010D, Q010R,Q019C, Q019D, Q019E, Q019H, Q019L, Q019P, Q019R, Q059A, Q059E, Q059L,Q059S, Q059T, Q103W, Q185D, Q185K, Q185R, Q185W, Q206G, Q206H, Q206L,Q206V, Q206W, Q217E, Q217F, Q217H, Q217L, Q217V, Q245M, Q271A, Q271D,Q271G, Q271L, Q271P, Q271T, Q271Y, Q275F, Q275L, Q275P, Q275R, S003D,S003F, S003K, S003R, S009K, S018D, S018R, S037A, S037G, S037K, S037L,S037P, S038M, S063A, S063F, S063G, S063M, S063R, S063Y, S087C, S087K,S087L, S087M, S087N, S087Y, S089A, S089D, S089F, S089G, S089H, S089I,S089K, S089R, S089V, S089Y, S130D, S130E, S130K, S130W, S145G, S145L,S145R, S145T, S159D, S159L, S159W, S161E, S161R, S162C, S162E, S162W,S163A, S182E, S182R, S183C, S183D, S183P, S183R, S188D, S188P, S204G,S204Y, S207G, S224G, S224T, S236C, S236G, S248D, S248H, S248R, S249E,S249L, S249R, S260A, S260G, S260K, S260Q, S260V, S260Y, T022L, T055D,T055E, T055I, T055K, T055M, T055S, T055V, T055Y, T158A, T158G, T158L,T158Q, T158V, T164K, T164Q, T208S, T244D, T244E, T244R, T253E, T253R,T253Y, T254G, T255D, T255E, T255K, T255R, V026A, V028I, V028L, V030I,V044C, V044P, V045E, V045G, V045N, V072L, V081A, V081G, V081H, V081S,V084I, V084M, V095A, V095C, V143A, V143F, V143H, V143Q, V143T, V143W,V147A, V147Q, V147S, V148I, V148L, V149C, V149I, V149L, V165L, V180A,V180C, V180M, V192C, V192F, V192I, V192Q, V192Y, V203A, V203G, V203K,V203S, V270C, V270L, V270P, W241F, Y006A, Y006M, Y006N, Y006R, Y006S,Y021C, Y091W, Y104V, Y104W, Y262C, Y262D, Y262E, Y262H, Y262I, Y262R,and Y262V, wherein amino acid positions of the variant are numbered bycorrespondence with the sequence of SEQ ID NO:2. Such variants may haveenhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or agreater PI value than BPN′ in this assay. The invention includes aprotease variant having proteolytic activity and/or a PI value of 0.9relative to BPN′-v36 in this BMI microswatch cleaning assay, the variantcomprising an amino acid sequence having at least 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ IDNO:2 or SEQ ID NO:6 and comprising at least one, two, three, four, five,six or more amino acid substitutions selected from said group above,wherein amino acid positions of the variant are numbered bycorrespondence with amino acid positions of the SEQ ID NO:2 sequence.Also included are compositions, including, but not limited to, e.g.,cleaning compositions, comprising at least one such variant and methodsfor cleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto or greater than 0.5 and less than 0.9 relative to BPN′-v36 in the BMImicroswatch cleaning assay of Test Method 3 in Detergent Composition 4at pH 8 and 16° C.: BPN′-S24G-S53G-S78N-S101N-G128A-Y217Q amino acidsequence (SEQ ID NO:6) comprising at least one amino acid substitutionselected from the group consisting of A001D, A001H, A001N, A015C, A048C,A048E, A085T, A133R, A137R, A142C, A144D, A144R, A152S, A153G, A187P,A187Q, A187T, A187V, A216R, A230S, A272R, A273H, A273T, A274H, D036N,D036S, D181H, D181T, D259N, D259P, D259S, E156G, E156H, E156L, E156Q,E156V, E251C, F189S, F189T, F189W, F189Y, F261E, G020C, G024D, G053M,G053R, G097R, G131D, G131R, G157N, G160R, G160V, G166L, G166W, G211E,G215D, G258A, G258D, G258P, I011T, I031C, I079E, I079R, I175L, I205C,K012H, K012N, K027A, K027N, K027S, K043A, K043D, K043E, K043G, K043L,K043M, K043P, K043V, K043W, K043Y, K136H, K141H, K141L, K141M, K141N,K141Q, K141T, K141V, K170G, K170S, K237T, K237V, K256D, K256S, K256T,K256V, K265N, K265S, L042F, L042M, L082E, L209A, L209E, L209G, L209R,L233G, L235V, L257D, L257E, L257P, L257R, L257W, L267E, M050L, N056D,N056S, N061C, N061D, N062A, N062H, N062L, N062V, N062Y, N076D, N076L,N076M, N078D, N078F, N101D, N101R, N118A, N212C, N212E, N218C, N218D,N218E, N252D, N252E, P014F, P014K, P057A, P057W, P172R, P194E, P201T,P210E, Q059C, Q059D, Q059R, Q185E, Q206D, Q217A, Q217K, Q217R, Q245A,Q245D, Q245E, Q245H, Q245R, Q271E, Q271F, Q271R, Q271W, Q275G, Q275I,R186I, R186L, R186V, R186W, S003E, S009C, S009E, S018C, S037D, S037E,S037H, S037R, S037Y, S038D, S038P, S038R, S038Y, S063L, S087D, S087R,S089C, S089E, S130C, S130R, S145D, S159C, S159P, S161C, S173T, S182C,S188E, S188F, S188K, S188L, S188R, S188W, S190A, S190G, S190T, S204R,S236D, S236E, S248C, S248E, S260C, S260E, S260R, T022P, T055C, T055W,T071A, T158D, T158E, T158P, T158R, T158Y, T164R, T242D, T242G, T255C,V004E, V004T, V045C, V045D, V045R, V045T, V051H, V081R, V143C, V143E,V143G, V192G, V203C, V203D, V203E, V203M, V203R, V270G, W241L, Y214H,Y214Q, A001E, A133E, A187L, A187N, A216C, A216H, A273Q, D099H, D259H,E156C, E195G, F189H, G131C, G146A, G166V, G215C, G215E, I107L, K012A,K012S, K012T, K043C, K170C, K256C, K256E, K265G, K265Y, L233E, M222F,M222S, N062Q, N076E, N078E, N184P, N218R, P005V, P014D, Q002K, Q002L,Q002R, Q010W, Q271C, R186H, S049C, S063C, S063D, S105T, S188C, S190C,S204E, T055R, T164G, V004D, V044T, V045I, V165C, V180S, Y006C, Y006D,Y006E, Y104T, A001C, A187C, A230G, A273D, A273P, D036Q, F189G, F189L,F189R, G157T, G178A, I031F, I111M, K012F, K012L, K027T, K043R, K136G,K141G, K170Q, M222A, M222L, N062R, N117G, N269C, P005W, P129V, P239A,P239H, P239T, Q059W, Q217G, Q275A, R186A, S191G, T164A, T220A, A001P,A187F, A187W, A273R, D041C, D060G, D197T, F189A, G046D, G157P, K012C,K012E, K012W, L042C, M222T, N062C, P239G, P239N, Q217C, R186M, S049T,S089P, S125A, S173V, and V044A, wherein amino acid positions of thevariant are numbered by correspondence with amino acid positions of theSEQ ID NO:2 sequence. Such variants have proteolytic activity. Theinvention includes a protease variant having proteolytic activity and/ora PI value equal to or greater than 0.5 and less than 0.9 relative toBPN′-v36 in this BMI microswatch cleaning assay(Test Method 3), thevariant comprising an amino acid sequence having at least 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity toSEQ ID NO:2 or SEQ ID NO:6 and comprising at least one, two, three,four, five, six or more amino acid substitutions selected from saidgroup above, wherein amino acid positions of the variant are numbered bycorrespondence with amino acid positions of the SEQ ID NO:2 sequence.Also included are compositions, including, but not limited to, e.g.,cleaning compositions, comprising at least one such variant and methodsfor cleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI valuegreater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from greater than 1.0 to about 10, from greater than1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36in an egg microswatch cleaning assay in Detergent Composition 4 at 16°C. and pH 8: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acidsequence (SEQ ID NO:6) comprising at least one amino acid substitutionselected from the group consisting of A216E, L090I, A098R, A098W, A098Y,A116G, A116R, A116S, A133M, I107L, I115V, M124L, N101I, N109H, N109S,N109T, N117R, P005G, Q185L, S089V, V095A, A015Y, A029G, A098D, A098E,A098G, A098N, A098S, A098T, A098V, A114S, A114T, A116E, A116L, A116T,A116V, A133H, A133L, A133S, A137G, A137I, A137L, A137S, A137V, A138S,A144S, A144V, A176S, A176T, A187T, A216F, A216P, A216Q, A216R, A216S,A216T, A216V, A216Y, D041E, D120A, D120E, D120Q, D120R, D120S, D181S,G020A, G020S, G024A, G097A, G097D, G097S, G131Q, G160S, G166I, G211L,G215N, H039N, H238N, I111L, I111V, I122A, L075I, L075Q, L135M, L209T,L209V, L233V, L235M, L235R, L257A, M119I, N025A, N025G, N025T, N061K,N101F, N101H, N101L, N101Q, N101R, N101S, N101T, N109A, N109G, N109K,N109L, N117E, N117H, N117K, N117S, N212G, N212S, N218F, N218G, N218H,N218L, N218S, N218W, N240Q, N252M, N252R, N252S, P005T, P040A, P040G,P040T, P129D, P129S, P194S, P210R, Q019R, Q019W, Q103L, Q103W, Q185A,Q185G, Q185M, Q185R, Q185T, Q206G, Q206Y, Q217A, Q217E, Q217R, Q217S,Q217T, S003Q, S009H, S018M, S033T, S130A, S130F, S130G, S130T, S130V,S145T, S159A, S161N, S161T, S162V, S162Y, S182L, S182W, S183F, S183L,S183V, S183W, S188K, S188W, S236Q, S236T, S248L, T022H, T022K, T208C,T253H, T255V, V044I, V121I, V139C, V143H, V143Q, V143T, V143W, V143Y,Y006K, Y021A, Y104W, A001F, A001G, A001H, A001K, A001L, A001Q, A001S,A001Y, A013V, A015G, A015K, A015R, A015S, A015T, A015W, A048S, A073N,A073S, A092S, A098K, A098P, A116D, A116W, A128S, A133P, A133T, A133V,A134G, A134S, A137H, A137N, A137T, A144D, A144K, A144L, A144M, A144N,A144R, A179G, A179S, A187V, A216G, A216L, A216W, A223S, A230C, A272K,A272L, A272P, A272S, A272T, A272W, A273G, A273S, A274G, A274M, A274T,D120K, D140E, D181A, D181E, D181G, D181H, D181T, D259E, D259N, D259Q,E054D, E156D, E156T, E251L, E251T, E251V, F058Y, F189W, F261K, F261Q,F261R, G007A, G007S, G020F, G020H, G020N, G020Q, G020T, G020Y, G024F,G024Q, G024R, G024T, G024V, G024W, G024Y, G053T, G097K, G097M, G097R,G097T, G131A, G131H, G131P, G131R, G131T, G131V, G160H, G160T, G166C,G166Q, G166S, G166T, G211A, G211D, G211K, G211M, G211N, G211Q, G211R,G211V, G211W, G215S, G215T, G215W, H017T, H017W, H017Y, H039V, H226A,H226F, H226I, H226L, H226M, H226V, I035V, I079A, I079S, I108V, I205V,I234L, I234V, I268V, K012S, K043P, K136H, K136R, K141A, K141F, K141T,K141W, K170A, K170G, K170R, K213A, K213R, K213S, K237A, K237H, K237L,K237S, K237V, K256A, K256G, K256H, K256M, K256P, K256Q, K256R, L016A,L016Q, L016T, L016V, L042V, L075M, L075T, L082M, L082V, L135F, L196I,L209H, L209Q, L209R, L209S, L209W, L233A, L233M, L233Q, L235I, L235K,L250I, L257S, L257T, L257V, L267A, L267Q, L267T, L267V, M119C, M199V,N025C, N025E, N025F, N025I, N025K, N025L, N025M, N025Q, N025V, N025Y,N061F, N061P, N061S, N061T, N076G, N078S, N101A, N109M, N109Q, N109R,N117A, N117M, N117Q, N118D, N118G, N118H, N118Q, N118R, N118S, N184A,N184C, N184G, N184L, N184R, N184S, N184T, N184V, N184W, N212C, N212F,N212I, N212K, N212L, N212P, N212Q, N212R, N212V, N212W, N212Y, N218A,N218P, N218T, N240A, N240E, N240G, N240H, N240L, N240R, N240S, N240T,N243C, N243Q, N243T, N243V, N252A, N252G, N252K, N252Q, P014G, P014Q,P014R, P014S, P014T, P040F, P040L, P040Q, P040S, P040V, P086C, P086H,P086S, P129A, P129E, P129G, P129K, P129R, P172A, P172K, P172Q, P172S,P194A, P194G, P194H, P194L, P194M, P194Q, P194R, P194V, P194W, P194Y,P210A, P210G, P210L, P210S, P239K, P239R, Q002A, Q002S, Q010A, Q010N,Q010R, Q010T, Q019A, Q019C, Q019D, Q019G, Q019S, Q019T, Q019V, Q059I,Q059V, Q103S, Q185F, Q185H, Q185I, Q185K, Q185N, Q185S, Q185Y, Q206H,Q206L, Q206P, Q206W, Q217F, Q217H, Q217I, Q217K, Q217L, Q217N, Q217V,Q271G, Q271R, Q271T, Q275F, Q275P, Q275R, R186A, R186I, R186K, S003A,S003F, S003G, S003H, S003K, S003R, S003T, S009T, S018N, S018T, S037G,S037T, S037V, S038G, S038Q, S063N, S063Q, S063T, S089M, S089N, S130K,S130L, S130R, S130W, S132N, S145G, S145K, S145M, S145R, S145V, S159C,S159H, S159L, S159Q, S159R, S159T, S159W, S161A, S161C, S161G, S161H,S161I, S161K, S161P, S161Q, S161R, S162F, S162G, S162I, S162L, S162M,S162N, S162P, S162R, S163G, S173A, S173G, S182F, S182G, S182K, S182N,S182Q, S182V, S183A, S183M, S183Q, S183R, S183T, S188A, S188F, S188G,S188P, S188R, S188T, S188V, S190C, S204A, S204G, S204I, S204L, S204Q,S204R, S204V, S207G, S224A, S224T, S236C, S236D, S236E, S236G, S236N,S248A, S248F, S248K, S248M, S248T, S249A, S249R, S249T, S249V, S249W,S249Y, S260G, S260H, S260K, S260N, T022A, T022G, T022Q, T022S, T022V,T022Y, T055A, T055K, T158A, T158S, T208L, T208S, T208V, T220S, T242D,T242N, T242S, T244E, T244G, T244I, T244R, T244V, T244W, T253A, T253G,T253N, T253S, T254S, T254V, T255H, T255I, T255K, T255L, T255Q, T255R,T255S, T255Y, V004A, V004N, V004P, V004W, V008A, V008M, V026I, V045S,V045W, V051I, V081Q, V081T, V084C, V093I, V095C, V143A, V143E, V143F,V143N, V143S, V147I, V147L, V147S, V147T, V148I, V149C, V149I, V165M,V180A, V180C, V180I, V180L, V180T, V192A, V192C, V192I, V192S, V192T,V192Y, V198L, V203A, V203F, V203K, V203L, V203M, V203N, V203Y, W241Y,Y006A, Y006G, Y006H, Y006L, Y006N, Y006P, Y006Q, Y006T, Y021E, Y021K,Y021L, Y021N, Y021Q, Y021R, Y021S, Y021T, Y104F, Y104I, Y104V, Y171F,Y214F, Y214L, Y214W, Y262F, and Y262S, wherein amino acid positions ofthe variant are numbered by correspondence with the sequence of SEQ IDNO:2. Such variants have enhanced proteolytic activity compared to BPN′(SEQ ID NO:2) and a greater PI value than BPN′ in this assay. Theinvention includes a protease variant having enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2), enhanced proteolytic activitycompared to BPN′-v3 and BPN′-v36, a PI value greater than that ofBPN′-v3, and/or a PI value of greater than 1.0 to about 5 relative toBPN′-v36 in this egg microswatch cleaning assay, the variant comprisingan amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 orSEQ ID NO:6 and comprising at least one, two, three, four, five, six ormore amino acid substitutions selected from said group above, whereinamino acid positions of the variant are numbered by correspondence withamino acid positions of the SEQ ID NO:2 sequence. Also included arecompositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto about 1.0 relative to BPN′-v36 in an egg microswatch cleaning assayin Detergent Composition 4 at pH 8 and 16° C.:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one amino acid substitution selected from thegroup consisting of BPN′-v36, A001D, A001M, A001N, A001R, A001T, A001V,A013C, A013G, A013S, A015D, A015F, A015L, A015M, A015P, A015Q, A074G,A074S, A085S, A085T, A085V, A088C, A088L, A088S, A088T, A088V, A133E,A133G, A133R, A137E, A144G, A144H, A144Q, A144T, A151C, A152S, A153G,A153S, A153V, A176C, A187G, A187Q, A187S, A200G, A228S, A228T, A230T,A230V, A231C, A231V, A232C, A232V, A272E, A272G, A272I, A272Q, A272R,A273L, A273V, A274L, A274Q, A274R, A274V, D036E, D259A, D259G, D259P,D259S, D259T, E156A, E156S, E156V, E195G, E251C, E251I, E251Q, F189Y,F261A, F261G, F261H, F261L, F261P, F261S, F261T, F261V, F261W, G020C,G020D, G020E, G020L, G020R, G024D, G024I, G024N, G024P, G024S, G053A,G053D, G053F, G053H, G053K, G053N, G053S, G053Y, G157A, G157S, G160A,G160L, G160P, G160R, G166A, G166L, G166V, G166W, G169A, G211E, G211P,G215A, G215D, G215E, G215H, G215V, G258D, G258S, H017F, H017I, H017L,H017M, H017V, H039C, H226S, H226Y, I011T, I011V, I031C, I031L, I031V,I079F, I079K, I079L, I079M, I079Q, I079R, I079T, I079V, I079W, I115L,I205A, I205C, I268L, K012R, K012T, K027R, K043A, K043E, K043F, K043H,K043I, K043M, K043N, K043Q, K043T, K043V, K043Y, K141H, K141Q, K141V,K170S, K213G, K213H, K213I, K213L, K213N, K213Q, K213T, K213W, K237I,K237N, K237R, K237T, K256C, K256E, K256S, K256T, K256V, K256W, K265H,K265R, L016E, L016F, L016I, L016S, L042I, L042M, L075A, L075H, L075Y,L082K, L082Q, L082T, L196M, L196V, L209A, L209C, L209E, L209G, L235W,L257C, L257H, L257Q, L257Y, L267E, L267F, L267R, L267S, M050Y, N025H,N025P, N025S, N056D, N061A, N061C, N061D, N061G, N061H, N061I, N061L,N061Q, N061R, N061V, N061W, N062A, N062S, N062V, N076A, N076E, N076L,N076M, N076Q, N076S, N076T, N076W, N078G, N078H, N078K, N078P, N078Q,N078T, N101D, N118A, N118T, N184E, N184H, N184I, N212A, N212D, N212E,N218C, N218D, N218E, N218M, N218R, N218V, N240W, N243A, N243G, N243P,N243S, N252D, N252E, N252L, N252T, N252V, N269S, P005A, P005D, P005M,P005Q, P014A, P014D, P014F, P014M, P014V, P040H, P040R, P040W, P040Y,P086A, P129T, P172E, P172G, P172R, P194E, P201A, P201G, P210E, P210V,Q002E, Q002G, Q010D, Q010F, Q010H, Q010I, Q010L, Q010S, Q019E, Q019H,Q019N, Q059A, Q059L, Q059R, Q059S, Q059T, Q185D, Q185E, Q206D, Q206S,Q206V, Q217G, Q245D, Q245E, Q245K, Q245M, Q245R, Q271A, Q271F, Q271P,Q271Y, Q275D, Q275H, Q275I, Q275L, Q275S, Q275W, R186H, R186L, R186V,R186W, S003D, S003E, S003M, S003P, S003V, S009A, S009E, S009G, S009I,S009K, S009M, S009P, S009W, S018A, S018G, S018I, S018L, S018P, S018R,S018V, S018W, S037A, S037D, S037Q, S037R, S037Y, S038D, S038H, S038K,S038M, S038R, S038T, S063A, S063G, S063K, S063M, S063R, S087A, S087D,S087F, S087G, S087Q, S087T, S089A, S089C, S089H, S089I, S089K, S089L,S089Q, S089R, S089T, S089Y, S130D, S130E, S145A, S145H, S145L, S159G,S161E, S161L, S161M, S161W, S162A, S162C, S162E, S162W, S163P, S173T,S182A, S182C, S182E, S182H, S182R, S182T, S183C, S183D, S183G, S183H,S188C, S188D, S188E, S188L, S190T, S191A, S204Y, S224C, S236A, S248C,S248D, S248G, S248I, S248N, S248Q, S248R, S248V, S249C, S249H, S249L,S249Q, S260A, S260C, S260E, S260P, S260Q, S260R, S260T, T022L, T022N,T022R, T055C, T055D, T055G, T055I, T055L, T055N, T055Q, T055S, T055V,T055Y, T071A, T071S, T158G, T158H, T158L, T158P, T158Q, T158R, T158V,T158Y, T164N, T164S, T244A, T244D, T244H, T244Q, T244S, T253Q, T253R,T253Y, T254A, T254G, T255A, T255D, T255E, V004E, V004G, V004R, V008C,V026A, V028L, V030I, V044C, V045D, V045E, V045H, V045M, V045Q, V045Y,V072L, V081L, V081S, V084A, V084I, V084M, V084T, V143C, V147C, V147Q,V149L, V165L, V180M, V192F, V192G, V192Q, V198I, V198M, V203C, V203E,V203H, V203I, V203Q, V203R, V203T, V203W, V270A, V270L, V270T, W241F,W241M, Y006C, Y006D, Y006E, Y006I, Y006M, Y006R, Y006S, Y006V, Y006W,Y021C, Y021D, Y021V, Y091W, Y167F, Y214V, Y262A, Y262C, Y262I, Y262M,Y262R, Y262T, Y262V, and Y262W, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have enhanced proteolytic activity compared to BPN′ (SEQID NO:2) and a greater PI value than BPN′ in this assay. The inventionincludes a protease variant having enhanced proteolytic activitycompared to BPN′ (SEQ ID NO:2), a PI value of about 1.0 relative toBPN′-v3, and/or a PI value of 1.0 relative to BPN′-v36 in this eggmicroswatch cleaning assay, the variant comprising an amino acidsequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 andcomprising at least one, two, three, four, five, six or more amino acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein. Thefollowing BPN′-v36 variants were determined to have a PI value equal toabout 0.9 relative to BPN′-v36 in an egg microswatch cleaning assay inDetergent Composition 4 at pH 8 and 16° C.:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one amino acid substitution selected from thegroup consisting of A001E, A015C, A015E, A048C, A048E, A073T, A085C,A085G, A088I, A088M, A114G, A137R, A187L, A187N, A187P, A187W, A216C,A230S, A273D, A273H, A273T, D036N, D099N, D259H, E156C, E156G, E156H,E156Q, F189H, F189R, F189S, F189T, F261C, F261D, F261E, G053E, G053M,G053Q, G131C, G131D, G157N, G160V, G215C, G215L, G258A, H039A, I011L,I079E, I268M, K012A, K012G, K012H, K012N, K027N, K043C, K043D, K043G,K043L, K043W, K136G, K141G, K141L, K141M, K141N, K141R, K170C, K170Q,K213V, K256D, K265G, K265N, K265Q, K265S, L082A, L082F, L082H, L082R,L082S, L090M, L233S, L235V, L257E, L257G, L257R, L257W, M222S, N025R,N062H, N062T, N076D, N076P, N078D, N078E, N078F, N078R, N078V, N269H,N269Q, P014K, P057A, P086F, P201T, Q002D, Q002I, Q002P, Q002V, Q019L,Q019P, Q059C, Q059D, Q059E, Q185W, Q271C, Q271D, Q271E, Q271L, Q271W,Q275G, R186M, S009C, S009L, S018D, S037E, S037H, S037K, S037L, S037P,S038P, S063C, S063D, S063F, S063L, S063Y, S087L, S087N, S087R, S087Y,S089D, S089F, S089G, S089W, S105T, S125A, S130C, S159D, S159P, S163A,S182P, S183P, S190A, S190G, S204E, S224G, S248E, S248H, S249E, S260V,S260Y, T055M, T055R, T055W, T158D, T158E, T164G, T164K, T164Q, T220A,T242G, T253E, T255C, T255G, V004D, V044L, V044P, V045C, V045G, V045L,V045N, V045R, V045V, V081A, V081G, V081H, V084S, V147A, V203D, V203G,V270C, V270P, V270S, W241L, Y104T, Y214Q, Y262D, Y262E, Y262G, Y262H,Y262L, Y262N, Y263G, and Y263W, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have proteolytic activity. The invention includes aprotease variant having enhanced proteolytic activity compared to BPN′(SEQ ID NO:2), and/or a PI value of 0.9 relative to BPN′-v36 in this eggmicroswatch cleaning assay, the variant comprising an amino acidsequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 andcomprising at least one, two, three, four, five, six or more amino acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto or greater than 0.5 and less than 0.9 relative to BPN′-v36 in an eggmicroswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one amino acid substitution selected from thegroup consisting of A001C, A142C, A187C, A216H, A273Q, A274H, D036Q,D036S, D099S, D197T, E156L, F189A, F189L, G053L, G053R, G157P, G178A,G258P, H039S, H238Y, K012C, K012E, K012L, K012W, K136E, K265Y, L075G,L075V, L082E, L126W, L257D, L257P, M050L, M222A, M222F, M222L, N056S,N062C, N062L, N062Y, N269C, P057W, Q002K, Q002L, Q217C, Q245A, Q245H,S018C, S038Y, S049C, S087C, S087K, S145D, S191G, T022P, T055E, T164A,T164R, V045K, V051H, V081R, V143G, V148L, V180S, V203S, V270G, Y214H,A187F, A273P, F189G, G046D, G146A, G157T, I031F, I175L, K012F, K027T,L042F, L233E, L233G, M222T, N062R, N184P, P005V, P005W, P129V, P239N,P239T, Q010W, Q059W, Q275A, V004T, V165C, A128H, A230G, D041C, H067T,K027S, K043R, L090T, N062Q, N117G, P225G, P225S, P239G, P239H, Q002R,S089E, V044A, V045I, A001P, A273R, D041N, D099A, D099H, D099Q, F058G,I111M, L042C, N118L, P239A, S049N, S089P, S173V, T242P, V044T, andV045T, wherein amino acid positions of the variant are numbered bycorrespondence with the sequence of SEQ ID NO:2. Such variants haveproteolytic activity. The invention includes a protease variant havingproteolytic activity and/or a PI value of equal to or greater than 0.5and less than 0.9 relative to BPN′-v36 in this egg microswatch cleaningassay, the variant comprising an amino acid sequence having at least60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one,two, three, four, five, six or more amino acid substitutions selectedfrom said group above, wherein amino acid positions of the variant arenumbered by correspondence with amino acid positions of the SEQ ID NO:2sequence. Also included are compositions, including, but not limited to,e.g., cleaning compositions, comprising at least one such variant andmethods for cleaning utilizing at least one such variant as described ingreater detail elsewhere herein.

Example 8 Cleaning Performance of Additional Combinatorial VariantsBased on BPN′-v36 Parent

Additional combinatorial variants based on parent BPN′-v36(BPN′-S24G-S53G-S78N-S101N-G128A-Y217Q) were made and provided by DNA2.0. These variants were tested for their cleaning performance using BMImicroswatch assay in Detergent Composition 4 at 16° C. and pH 8, BMImicroswatch assay in Detergent Composition 4 at 16° C. and pH 7, Eggmicroswatch assay in Detergent Composition 4 at 16° C. and pH 8, andGrass microswatch assay in Detergent Composition 4 at 16° C. and pH 8.Protein content was determined using TCA assay and protease activity wasassayed using AAPF assay. All assays were performed as described inExample 1 and the Performance Indices were calculated relative toBPN′-v36.

The following BPN′-v36 variants were determined to have a PI valuegreater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from greater than 1.0 to about 10, from greater than1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence(SEQ ID NO:6) comprising at least one set of amino acid substitutionsselected from the group consisting of A088T-L257G, A116T-A128S,N061S-N109G-A128S-N243V-S260P, S009T-N109G-A128S-K141R-N243V,S009T-S018T-Y021N-N109G-A128S-K141R, and S162G-K256R, wherein amino acidpositions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. Such variants have enhanced proteolyticactivity compared to BPN′, BPN′-v3, and BPN′-v36, and a greater PI valuethan BPN′, BPN′-v3 and BPN′-v36 in this assay. The invention includes aprotease variant having enhanced proteolytic activity compared to BPN′,BPN′-v3, and BPN′-v36, a PI value of greater than 1.0 to about 5relative to BPN′-v3, and/or a PI value of greater than 1.0 to about 5relative to BPN′-v36 in this BMI microswatch cleaning assay, the variantcomprising an amino acid sequence having at least 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ IDNO:2 or SEQ ID NO:6 and comprising at least one set of amino acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence.

The following BPN′-v36 variants were determined to have a PI value equalto about 1.0 relative to BPN′-v36 in a BMI microswatch cleaning assay inDetergent Composition 4 at pH 8 and 16° C.:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of BPN′-v36, A088T, A088T-A116T, A088T-G131H,A088T-K256R, A088T-N109G, A088T-N243V, A088T-Q103H, A088T-S162G,A088T-S248N, A088T-S249A, A088T-T158S, A116T, A116T-G131H, A116T-K256R,A116T-L257G, A116T-N243V, A116T-S162G, A116T-S248N, A116T-S249A,A116T-T158S, A128S-K256R, A128S-L257G, A128S-N243V-S248N-K256R,A128S-S162G, A128S-S248N, A128S-S249A, A128S-T158S, G024E-A116T,G024E-K256R, G024E-L257G, G024E-N109G, G024E-N243V, G024E-T158S, G131H,G131H-K256R, G131H-L257G, G131H-N243V-K256R, G131H-S162G, G131H-S248N,G131H-S249A, G131H-T158S, K043Y-A088T, K043Y-K256R, K043Y-N243V, K256R,K256R-L257G, L257G, N061G-N109G-N243V, N061P-N109G-G131H-N243V,N061P-N109G-N243V, N061S-A128S-N243V-S260P,N061S-N109G-A128S-N243V-S248N-K256R-S260P, N061S-N109G-A128S-S260P,N061S-N109G-N243V, N076D-K256R, N076D-L257G, N076D-N109G, N076D-T158S,N109A-A128S-N243V-K256R, N109G, N109G-A116T, N109G-A128S,N109G-A128S-G131H-N243V-S248N-K256R, N109G-A128S-N243V-K256R,N109G-A128S-N243V-S248A, N109G-A128S-N243V-S248A-K256R,N109G-A128S-N243V-S248N, N109G-A128S-N243V-S248N-K256R,N109G-A128S-N243V-S248N-K256R-L257G,N109G-A128S-S162G-N243V-S248N-K256R, N109G-A128S-S248N-K256R,N109G-A128S-T158S-N243V-S248N-K256R, N109G-G131H, N109G-K256R,N109G-L257G, N109G-N218S, N109G-N243P-S248A-K256R,N109G-N243P-S248N-K256R, N109G-N243V, N109G-N243V-K256R,N109G-N243V-S248A-K256R, N109G-N243V-S248N, N109G-N243V-S248N-K256R,N109G-S162G, N109G-S248N-K256R, N109G-S249A, N109G-T158S,N109Q-A128S-N243V-K256R, N109S-A128S-N243V-K256R, N218S-N243V, N243V,N243V-K256R, N243V-L257G, N243V-S248N, N243V-S248N-K256R, N243V-S249A,P040A-N109G-A128S-N243V-S248N-K256R, Q103H-A116T, Q103H-A128S,Q103H-G131H, Q103H-K256R, Q103H-L257G, Q103H-N109G, Q103H-N218S,Q103H-N243V, Q103H-S162G, Q103H-S248N, Q103H-S249A, Q103H-T158S,S009T-A128S-K141R-N243V, S009T-N109G-A128S-K141R,S009T-N109G-A128S-K141R-N243V-S248N-K256R,S009T-S018T-Y021N-A128S-K141R-N243V, S018T-Y021N-A128S-N243V,S018T-Y021N-N061S-A128S-N243V-S260P,S018T-Y021N-N061S-N109G-A128S-S260P, S018T-Y021N-N109G-A128S,S018T-Y021N-N109G-A128S-N243V,S018T-Y021N-N109G-A128S-N243V-S248N-K256R,S033T-N109G-A128S-N243P-S248N-K256R, S033T-N243V, S033T-Q103H,S033T-T158S, S063G, S063G-A088T, S063G-A128S, S063G-K256R, S063G-L257G,S063G-N076D, S063G-N109G, S063G-Q103H, S063G-S162G, S063G-S248N,S063G-T158S, S162G, S162G-L257G, S162G-N243V, S162G-S248N, S248N,S248N-L257G, S249A, T158S, T158S-L257G, T158S-N218S, T158S-N243V,T158S-S248N, and T158S-S249A, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have enhanced proteolytic activity compared to BPN′ (SEQID NO:2) and a greater PI value than BPN′ in this assay. The inventionincludes a protease variant having enhanced proteolytic activitycompared to BPN′ (SEQ ID NO:2), a PI value of 1.0 relative to BPN′-v3,and a PI value of 1.0 relative to BPN′-v36 in this BMI microswatchcleaning assay Test Method 3), the variant comprising an amino acidsequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 andcomprising at least one set of amino acid substitutions selected fromsaid group above, wherein amino acid positions of the variant arenumbered by correspondence with amino acid positions of the SEQ ID NO:2sequence. Also included are compositions, including, but not limited to,e.g., cleaning compositions, comprising at least one such variant andmethods for cleaning utilizing at least one such variant as described ingreater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto about 0.9 relative to BPN′-v36 in a BMI microswatch cleaningassay(test Method 3) in Detergent Composition 4 at pH 8 and 16° C.:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of A001E-A088T, A001E-A116T,A001E-A128S-G131H-N243V, A001E-G131H-G169A-N243V, A001E-K256R,A001E-N109G, A001E-N243V, A001E-S033T, A001E-S033T-N109G-N218S,A001E-S033T-N109G-N243V, A001E-S162G, A001E-T158S, A088T-A128S,A088T-G169A, A088T-N218S, A088T-Q206D, A116T-G169A, A116T-N218S,A116T-Q206D, A128S, A128S-G131H, A128S-G169A, A128S-N218S, A128S-N243V,A128S-Q206D, G024E, G024E-A088T, G024E-A128S, G024E-G131H, G024E-K043Y,G024E-N218S, G024E-Q103H, G024E-S033T, G024E-S063G, G024E-S162G,G024E-S248N, G024E-S249A, G131H-G169A, G131H-N218S, G131H-N243V,G131H-Q206D, G169A, G169A-K256R, G169A-L257G, G169A-N218S, G169A-N243V,G169A-Q206D, G169A-S248N, G169A-S249A, K043Y, K043Y-A116T, K043Y-A128S,K043Y-G131H, K043Y-G169A, K043Y-L257G, K043Y-N109G, K043Y-N218S,K043Y-Q103H, K043Y-S063G, K043Y-S162G, K043Y-S248N, K043Y-S249A,K043Y-T158S, N076D, N076D-A088T, N076D-A128S, N076D-G131H, N076D-N218S,N076D-N243V, N076D-Q103H, N076D-S162G, N076D-S248N, N076D-S249A,N109G-G169A, N109G-Q206D, N109G-S248N, N218S, N218S-K256R, N218S-L257G,N218S-S248N, N218S-S249A, P040E-N109G-A128S-G131H, Q103H, Q103H-G169A,Q206D, Q206D-K256R, Q206D-L257G, Q206D-N218S, Q206D-N243V, Q206D-S248N,Q206D-S249A, S018T-Y021N-S033T-N109G-A128S-N243V-S248N-K256R, S033T,S033T-A088T, S033T-A116T, S033T-A128S, S033T-A128S-G131H-N243P,S033T-G131H, S033T-K043Y, S033T-K256R, S033T-L257G, S033T-N076D,S033T-N076D-A128S-N218S,S033T-N076D-N109G-A128S-N218S-N243V-S248N-K256R, S033T-N109G,S033T-N109G-A128S-N243V-S248N-K256R, S033T-N218S,S033T-P040E-Q103H-N109G, S033T-Q103H-A128S-G131H, S033T-Q206D,S033T-S063G, S033T-S162G, S033T-S248N, S033T-S249A, S063G-A116T,S063G-G131H, S063G-G169A, S063G-N109G-A128S-G131H, S063G-N218S,S063G-N243V, S063G-Q206D, S063G-S249A, S162G-G169A, S162G-N218S,S162G-Q206D, S162G-S249A, S248N-K256R, S248N-S249A, S249A-K256R,S249A-L257G, T158S-G169A, T158S-K256R, T158S-Q206D, and T158S-S162G,wherein amino acid positions of the variant are numbered bycorrespondence with the sequence of SEQ ID NO:2. Such variants haveproteolytic activity. The invention includes a protease variant havingenhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), and/or aPI value of 0.9 relative to BPN′-v36 in this BMI microswatch cleaningassay, the variant comprising an amino acid sequence having at least60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least oneset of acid substitutions selected from said group above, wherein aminoacid positions of the variant are numbered by correspondence with aminoacid positions of the SEQ ID NO:2 sequence. Also included arecompositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto or greater than 0.5 and less than 0.9 relative to BPN′-v36 in a BMImicroswatch cleaning assay (Test Method 3) in Detergent Composition 4 atpH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acidsequence (SEQ ID NO:6) comprising at least one set of amino acidsubstitutions selected from the group consisting of A001E, A001E-A128S,A001E-G024E, A001E-G131H, A001E-G169A, A001E-L257G, A001E-N218S,A001E-Q103H, A001E-S063G, A001E-S248N, A001E-S249A, G024E-N076D,K043Y-N076D, K043Y-Q206D, N076D-A116T, N076D-G169A, N076D-Q206D,Q103H-Q206D, S033T-G169A,S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243P,S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243V, A001E-K043Y,A001E-N076D, A001E-N076D-N109G-A128S, A001E-Q206D, and G024E-Q206D,wherein amino acid positions of the variant are numbered bycorrespondence with the sequence of SEQ ID NO:2. Such variants haveproteolytic activity. The invention includes a protease variant havingproteolytic activity and/or a PI value equal to or greater than 0.5 andless than 0.9 relative to BPN′-v36 in this BMI microswatch cleaningassay, the variant comprising an amino acid sequence having at least60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least oneset of acid substitutions selected from said group above, wherein aminoacid positions of the variant are numbered by correspondence with aminoacid positions of the SEQ ID NO:2 sequence. Also included arecompositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI valuegreater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from greater than 1.0 to about 10, from greater than1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36in a BMI microswatch cleaning assay (test Method 3) in DetergentComposition 4 at pH 7 and 16° C.:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of A116T, A088T-N243V, G024E-A116T, K043Y,N076D-A116T, N218S-S248N, S033T-N243V, S033T-S063G, S248N-L257G,A001E-S249A, A088T-A116T, A088T-A128S, A088T-G131H, A088T-L257G,A088T-N109G, A088T-S248N, A088T-S249A, A116T-N243V, A116T-T158S, A128S,A128S-K256R, A128S-L257G, A128S-N243V, A128S-S248N, A128S-T158S,G024E-A088T, G024E-A128S, G024E-G131H, G024E-K256R, G024E-L257G,G024E-N218S, G024E-N243V, G024E-S162G, G024E-S249A, G024E-T158S, G131H,G131H-K256R, G131H-S249A, K043Y-A088T, K043Y-A116T, K256R, N076D-K256R,N109G, N109G-A116T, N109G-A128S, N109G-A128S-N243V-K256R,N109G-A128S-N243V-S248A, N109G-G131H, N109G-K256R, N109G-L257G,N109G-N218S, N109G-N243V, N109G-S248N, N218S-L257G, N243V, N243V-K256R,N243V-L257G, N243V-S248N, N243V-S249A, Q103H-A128S, Q103H-G131H,Q103H-K256R, Q103H-L257G, Q103H-N243V, Q103H-S248N, Q103H-S249A,Q103H-T158S, Q206D-N243V, S033T-A128S, S033T-K256R, S033T-N076D,S033T-N218S, S033T-S248N, S033T-T158S, S063G-A128S, S063G-K256R,S063G-N243V, S063G-S162G, S063G-T158S, S162G-K256R, S248N-K256R, S249A,T158S-N243V, and T158S-S249A, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have enhanced proteolytic activity compared to BPN′,BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3 andBPN′-v36 in this assay. The invention includes a protease variant havingenhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), enhancedproteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, a PI valueof greater than 1.0 to about 5 relative to BPN′-v3, and/or a PI value ofgreater than 1.0 to about 5 relative to BPN′-v36 in this BMI microswatchcleaning assay, the variant comprising an amino acid sequence having atleast 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at leastone set of amino acid substitutions selected from said group above,wherein amino acid positions of the variant are numbered bycorrespondence with amino acid positions of the SEQ ID NO:2 sequence.Also included are compositions, including, but not limited to, e.g.,cleaning compositions, comprising at least one such variant and methodsfor cleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto about 1.0 relative to BPN′-v36 in a BMI microswatch cleaningassay(Test Method 3) in Detergent Composition 4 at pH 7 and 16° C.:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of BPN′-v36, A001E-A128S, A001E-G131H,A001E-K256R, A001E-N218S, A001E-N243V, A001E-S033T, A001E-S063G,A001E-S162G, A088T, A088T-K256R, A088T-N218S, A088T-Q103H, A088T-S162G,A088T-T158S, A116T-A128S, A116T-G131H, A116T-K256R, A116T-L257G,A116T-S162G, A116T-S248N, A116T-S249A, A128S-G169A, A128S-N218S,A128S-S162G, A128S-S249A, G024E, G024E-N109G, G024E-Q103H, G024E-S033T,G024E-S063G, G024E-S248N, G131H-L257G, G131H-N243V, G131H-S162G,G131H-T158S, G169A, G169A-L257G, G169A-S248N, K043Y-A128S, K043Y-G131H,K043Y-K256R, K043Y-L257G, K043Y-N109G, K043Y-N243V, K043Y-Q103H,K043Y-S063G, K043Y-S162G, K043Y-S248N, K043Y-S249A, K043Y-T158S,K256R-L257G, L257G, N061G-N109G-N243V, N061S-A128S-N243V-S260P,N061S-N109G-A128S-N243V-S260P, N061S-N109G-A128S-S260P, N076D-A088T,N076D-A128S, N076D-G169A, N076D-N218S, N076D-N243V, N076D-S162G,N076D-S248N, N076D-T158S, N109A-A128S-N243V-K256R,N109G-A128S-G131H-N243V-S248N-K256R, N109G-A128S-N243V-S248A-K256R,N109G-A128S-N243V-S248N-K256R-L257G,N109G-A128S-S162G-N243V-S248N-K256R,N109G-A128S-T158S-N243V-S248N-K256R, N109G-Q206D, N109G-S162G,N109G-S249A, N109G-T158S, N109Q-A128S-N243V-K256R,N109S-A128S-N243V-K256R, N218S, N218S-K256R, N218S-N243V,P040A-N109G-A128S-N243V-S248N-K256R, Q103H, Q103H-A116T, Q103H-G169A,Q103H-N109G, Q103H-N218S, Q103H-S162G, S009T-A128S-K141R-N243V,S009T-N109G-A128S-K141R, S009T-N109G-A128S-K141R-N243V,S009T-S018T-Y021N-N109G-A128S-K141R, S018T-Y021N-N109G-A128S,S018T-Y021N-N109G-A128S-N243V,S018T-Y021N-N109G-A128S-N243V-S248N-K256R, S033T-A088T, S033T-A116T,S033T-G131H, S033T-K043Y, S033T-L257G, S033T-N109G, S033T-Q103H,S033T-Q206D, S033T-S162G, S033T-S249A, S063G, S063G-A088T, S063G-A116T,S063G-L257G, S063G-N076D, S063G-N109G, S063G-N218S, S063G-Q103H,S063G-S248N, S063G-S249A, S162G, S162G-G169A, S162G-L257G, S162G-N218S,S162G-N243V, S162G-S248N, S162G-S249A, S248N, S248N-S249A, S249A-K256R,S249A-L257G, T158S, T158S-G169A, T158S-K256R, T158S-L257G, T158S-N218S,and T158S-S248N, wherein amino acid positions of the variant arenumbered by correspondence with the sequence of SEQ ID NO:2. Suchvariants have enhanced proteolytic activity compared to BPN′ protease(SEQ ID NO:2) and a greater PI value than BPN′ in this assay. Theinvention includes a protease variant having enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2), a PI value of 1.0 relative toBPN′-v3, and a PI value of 1.0 relative to BPN′-v36 in this BMImicroswatch cleaning assay, the variant comprising an amino acidsequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 andcomprising at least one set of amino acid substitutions selected fromsaid group above, wherein amino acid positions of the variant arenumbered by correspondence with amino acid positions of the SEQ ID NO:2sequence. Also included are compositions, including, but not limited to,e.g., cleaning compositions, comprising at least one such variant andmethods for cleaning utilizing at least one such variant as described ingreater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto about 0.9 relative to BPN′-v36 in a BMI microswatch cleaning assay(Test Method 3) in Detergent Composition 4 at pH 7 and 16° C.:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of A001E, A001E-A088T, A001E-A116T,A001E-G169A, A001E-L257G, A001E-N109G, A001E-S033T-N109G-N243V,A001E-T158S, A088T-G169A, A088T-Q206D, A116T-N218S, A128S-G131H,A128S-N243V-S248N-K256R, A128S-Q206D, G024E-K043Y, G024E-N076D,G024E-Q206D, G131H-G169A, G131H-N218S, G131H-N243V-K256R, G131H-Q206D,G131H-S248N, G169A-K256R, G169A-N218S, G169A-N243V, G169A-Q206D,K043Y-N076D, K043Y-N218S, N061P-N109G-G131H-N243V, N061P-N109G-N243V,N061S-N109G-A128S-N243V-S248N-K256R-S260P, N061S-N109G-N243V, N076D,N076D-G131H, N076D-L257G, N076D-N109G, N076D-Q103H, N076D-S249A,N109G-A128S-N243V-S248N, N109G-A128S-N243V-S248N-K256R,N109G-A128S-S248N-K256R, N109G-N243P-S248A-K256R,N109G-N243P-S248N-K256R, N109G-N243V-K256R, N109G-N243V-S248A-K256R,N109G-N243V-S248N, N109G-N243V-S248N-K256R, N109G-S248N-K256R,N218S-S249A, N243V-S248N-K256R, Q103H-Q206D, Q206D, Q206D-K256R,Q206D-N218S, Q206D-S248N, Q206D-S249A,S009T-N109G-A128S-K141R-N243V-S248N-K256R,S009T-S018T-Y021N-A128S-K141R-N243V, S018T-Y021N-A128S-N243V,S018T-Y021N-N061S-A128S-N243V-S260P,S018T-Y021N-N061S-N109G-A128S-S260P,S018T-Y021N-S033T-N109G-A128S-N243V-S248N-K256R, S033T, S033T-G169A,S033T-N076D-A128S-N218S,S033T-N076D-N109G-A128S-N218S-N243V-S248N-K256R,S033T-N109G-A128S-N243P-S248N-K256R,S033T-N109G-A128S-N243V-S248N-K256R, S063G-G131H, S063G-G169A,S162G-Q206D, and T158S-S162G, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have proteolytic activity. The invention includes aprotease variant having proteolytic activity and/or a PI value of 0.9relative to BPN′-v36 in this BMI microswatch cleaning assay, the variantcomprising an amino acid sequence having at least 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ IDNO:2 or SEQ ID NO:6 and comprising at least one set of acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto or greater than 0.5 and less than 0.9 relative to BPN′-v36 in a BMImicroswatch cleaning assay in Detergent Composition 4 at pH 7 and 16°C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of A001E-A128S-G131H-N243V, A001E-G024E,A001E-G131H-G169A-N243V, A001E-Q103H, A001E-S033T-N109G-N218S,A001E-S248N, A116T-G169A, A116T-Q206D, G169A-S249A, K043Y-G169A,N109G-G169A, P040E-N109G-A128S-G131H, Q206D-L257G,S033T-A128S-G131H-N243P, S033T-A128S-G131H-N243V,S033T-P040E-Q103H-N109G, S033T-Q103H-A128S-G131H,S063G-N109G-A128S-G131H, S063G-Q206D, T158S-Q206D, A001E-K043Y,A001E-N076D, A001E-Q206D,S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243P,S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243V,A001E-N076D-N109G-A128S, K043Y-Q206D, and N076D-Q206D, wherein aminoacid positions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. Such variants have proteolytic activity. Theinvention includes a protease variant having proteolytic activity and/ora PI value equal to or greater than 0.5 and less than 0.9 relative toBPN′-v36 in this assay, the variant comprising an amino acid sequencehaving at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprisingat least one set of acid substitutions selected from said group above,wherein amino acid positions of the variant are numbered bycorrespondence with amino acid positions of the SEQ ID NO:2 sequence.Also included are compositions, including, but not limited to, e.g.,cleaning compositions, comprising at least one such variant and methodsfor cleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI valuegreater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from greater than 1.0 to about 10, from greater than1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence(SEQ ID NO:6) comprising at least one set of amino acid substitutionsselected from the group consisting of A088T-L257G, G024E-K256R,G024E-L257G, N109G-A116T, N109G-L257G, N243V-K256R, S033T-N109G,S033T-T158S, S063G-L257G, A001E-L257G, A088T-A128S, A088T-G169A,A088T-K256R, A088T-N109G, A088T-N218S, A088T-N243V, A088T-S248N,A088T-T158S, A116T, A116T-A128S, A116T-G131H, A116T-K256R, A116T-L257G,A116T-N218S, A116T-S162G, A116T-T158S, A128S, A128S-G169A, A128S-K256R,A128S-L257G, A128S-N218S, G024E, G024E-A128S, G024E-G131H, G024E-N109G,G024E-N243V, G024E-S033T, G024E-S063G, G024E-S248N, G024E-S249A,G024E-T158S, G131H, G131H-G169A, G131H-K256R, G131H-N218S, G131H-S249A,G169A, G169A-L257G, G169A-N243V, K043Y-A088T, K043Y-N109G, K256R,K256R-L257G, N061G-N109G-N243V, N076D-N109G, N109G, N109G-A128S,N109G-G131H, N109G-K256R, N109G-N218S, N109G-S162G, N109G-S248N,N109G-S249A, N109G-T158S, N218S, N218S-K256R, N218S-L257G, N218S-S248N,N243V, N243V-L257G, N243V-S248N, N243V-S249A,P040A-N109G-A128S-N243V-S248N-K256R, Q103H-K256R, Q103H-L257G,Q103H-N109G, S009T-S018T-Y021N-N109G-A128S-K141R, S033T-A088T,S033T-A116T, S033T-A128S, S033T-G131H, S033T-K043Y, S033T-K256R,S033T-L257G, S033T-N076D, S033T-N218S, S033T-N243V, S033T-Q103H,S033T-S063G, S033T-S162G, S033T-S248N, S033T-S249A, S063G, S063G-A088T,S063G-A116T, S063G-A128S, S063G-G131H, S063G-K256R, S063G-N109G,S063G-N218S, S063G-N243V, S063G-S248N, S063G-S249A, S063G-T158S,S162G-K256R, S162G-N218S, S162G-N243V, S162G-S248N, S162G-S249A, S248N,S249A, S249A-L257G, T158S, T158S-L257G, and T158S-N243V, wherein aminoacid positions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. Such variants have enhanced proteolyticactivity compared to BPN′, BPN′-v3, and BPN′-v36, and a greater PI valuethan BPN′, BPN′-v3 and BPN′-v36 in this assay. The invention includes aprotease variant having enhanced proteolytic activity compared to BPN′(SEQ ID NO:2), enhanced proteolytic activity compared to BPN′, BPN′-v3,and BPN′-v36, a PI value of greater than 1.0 to about 5 relative toBPN′-v3, and/or a PI value of greater than 1.0 to about 5 relative toBPN′-v36 in this egg microswatch cleaning assay, the variant comprisingan amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 orSEQ ID NO:6 and comprising at least one set of amino acid substitutionsselected from said group above, wherein amino acid positions of thevariant are numbered by correspondence with amino acid positions of theSEQ ID NO:2 sequence. Also included are compositions, including, but notlimited to, e.g., cleaning compositions, comprising at least one suchvariant and methods for cleaning utilizing at least one such variant asdescribed in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto about 1.0 relative to BPN′-v36 in an egg microswatch cleaning assayin Detergent Composition 4 at pH 8 and 16° C.:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of BPN′-v36, A001E, A001E-A116T, A001E-G131H,A001E-G169A, A001E-K256R, A001E-N109G, A001E-S033T-N109G-N243V,A001E-S063G, A001E-S248N, A001E-S249A, A001E-T158S, A088T, A088T-A116T,A088T-G131H, A088T-Q103H, A088T-Q206D, A088T-S162G, A088T-S249A,A116T-G169A, A116T-N243V, A116T-S248N, A116T-S249A, A128S-G131H,A128S-N243V, A128S-S162G, A128S-S248N, A128S-S249A, A128S-T158S,G024E-A088T, G024E-A116T, G024E-K043Y, G024E-N076D, G024E-N218S,G024E-Q103H, G024E-S162G, G131H-L257G, G131H-N243V, G131H-N243V-K256R,G131H-S162G, G131H-S248N, G131H-T158S, G169A-K256R, G169A-N218S,G169A-Q206D, G169A-S248N, G169A-S249A, K043Y, K043Y-A116T, K043Y-A128S,K043Y-G169A, K043Y-K256R, K043Y-L257G, K043Y-N076D, K043Y-N218S,K043Y-N243V, K043Y-S063G, K043Y-S248N, K043Y-S249A, K043Y-T158S, L257G,N061P-N109G-G131H-N243V, N061P-N109G-N243V, N061S-A128S-N243V-S260P,N061S-N109G-A128S-N243V-S248N-K256R-S260P, N061S-N109G-A128S-S260P,N061S-N109G-N243V, N076D, N076D-A088T, N076D-A116T, N076D-G131H,N076D-G169A, N076D-K256R, N076D-L257G, N076D-N218S, N076D-N243V,N076D-Q103H, N076D-S249A, N076D-T158S, N109A-A128S-N243V-K256R,N109G-A128S-G131H-N243V-S248N-K256R, N109G-A128S-N243V-K256R,N109G-A128S-N243V-S248A, N109G-A128S-N243V-S248A-K256R,N109G-A128S-N243V-S248N, N109G-A128S-N243V-S248N-K256R,N109G-A128S-N243V-S248N-K256R-L257G,N109G-A128S-S162G-N243V-S248N-K256R, N109G-G169A,N109G-N243P-S248A-K256R, N109G-N243V-K256R, N109G-N243V-S248A-K256R,N109G-N243V-S248N, N109G-N243V-S248N-K256R, N109G-S248N-K256R,N109Q-A128S-N243V-K256R, N109S-A128S-N243V-K256R, N218S-N243V,N218S-S249A, Q103H, Q103H-A116T, Q103H-A128S, Q103H-G131H, Q103H-G169A,Q103H-N218S, Q103H-N243V, Q103H-S162G, Q103H-S248N, Q103H-S249A,Q103H-T158S, Q206D, Q206D-L257G, Q206D-N218S, S009T-A128S-K141R-N243V,S009T-N109G-A128S-K141R, S009T-N109G-A128S-K141R-N243V,S009T-N109G-A128S-K141R-N243V-S248N-K256R,S009T-S018T-Y021N-A128S-K141R-N243V, S018T-Y021N-A128S-N243V,S018T-Y021N-N109G-A128S, S018T-Y021N-N109G-A128S-N243V,S018T-Y021N-N109G-A128S-N243V-S248N-K256R,S018T-Y021N-S033T-N109G-A128S-N243V-S248N-K256R, S033T,S033T-A128S-G131H-N243V, S033T-G169A,S033T-N109G-A128S-N243P-S248N-K256R,S033T-N109G-A128S-N243V-S248N-K256R, S033T-Q103H-A128S-G131H,S033T-Q206D, S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243V,S063G-G169A, S063G-N076D, S063G-N109G-A128S-G131H, S063G-Q103H,S063G-S162G, S162G, S162G-G169A, S162G-L257G, S248N-K256R, S248N-L257G,S248N-S249A, S249A-K256R, T158S-G169A, T158S-K256R, T158S-N218S,T158S-S162G, T158S-S248N, and T158S-S249A, wherein amino acid positionsof the variant are numbered by correspondence with the sequence of SEQID NO:2. Such variants have enhanced proteolytic activity compared toBPN′ protease (SEQ ID NO:2) and a greater PI value than BPN′ in thisassay. The invention includes a protease variant having enhancedproteolytic activity compared to BPN′ (SEQ ID NO:2), a PI value of 1.0relative to BPN′-v3, and a PI value of 1.0 relative to BPN′-v36 in thisegg microswatch cleaning assay, the variant comprising an amino acidsequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 andcomprising at least one set of amino acid substitutions selected fromsaid group above, wherein amino acid positions of the variant arenumbered by correspondence with amino acid positions of the SEQ ID NO:2sequence. Also included are compositions, including, but not limited to,e.g., cleaning compositions, comprising at least one such variant andmethods for cleaning utilizing at least one such variant as described ingreater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto or greater than 0.5 and less than 0.9 relative to BPN′-v36 in an eggmicroswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of A001E-A088T, A001E-A128S,A001E-A128S-G131H-N243V, A001E-G024E, A001E-G024E-S204E-Q206D,A001E-G131H-G169A-N243V, A001E-K043Y, A001E-N076D,A001E-N076D-N109G-A128S, A001E-N218S, A001E-N243V, A001E-Q103H,A001E-Q206D, A001E-S033T, A001E-S033T-N109G-N218S, A001E-S162G,A116T-Q206D, A128S-N243V-S248N-K256R, A128S-Q206D, G024E-Q206D,G131H-Q206D, K043Y-G131H, K043Y-Q103H, K043Y-Q206D, K043Y-S162G,N061S-N109G-A128S-N243V-S260P, N076D-A128S, N076D-Q206D, N076D-S162G,N076D-S248N, N109G-A128S-S248N-K256R,N109G-A128S-T158S-N243V-S248N-K256R, N109G-N243P-S248N-K256R,N109G-Q206D, N243V-S248N-K256R, P040E-N109G-A128S-G131H, Q103H-Q206D,Q206D-K256R, Q206D-N243V, Q206D-S248N, Q206D-S249A,S018T-Y021N-N061S-A128S-N243V-S260P,S018T-Y021N-N061S-N109G-A128S-S260P, S033T-A128S-G131H-N243P,S033T-N076D-A128S-N218S,S033T-N076D-N109G-A128S-N218S-N243V-S248N-K256R,S033T-P040E-Q103H-N109G,S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243P, S063G-Q206D,S162G-Q206D, and T158S-Q206D, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have proteolytic activity. The invention includes aprotease variant having proteolytic activity and/or a PI value equal toor greater than 0.5 and less than 0.9 relative to BPN′-v36 in thisassay, the variant comprising an amino acid sequence having at least60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least oneset of acid substitutions selected from said group above, wherein aminoacid positions of the variant are numbered by correspondence with aminoacid positions of the SEQ ID NO:2 sequence. Also included arecompositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI valuegreater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from greater than 1.0 to about 10, from greater than1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36in a grass microswatch cleaning assay in Detergent Composition 4 at pH 8and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence(SEQ ID NO:6) comprising at least one set of amino acid substitutionsselected from the group consisting of T158S-L257G, K256R, L257G,S033T-N109G, S162G-K256R, S162G-L257G, G024E-K256R, G024E-L257G,G024E-S033T, N109G-A116T, N218S-L257G, S033T-A088T, S033T-A116T,S033T-N243V, S033T-Q103H, S162G-N218S, S162G-N243V, T158S, T158S-N218S,T158S-N243V, A088T, A088T-G169A, A088T-K256R, A088T-L257G, A088T-S162G,A088T-T158S, A116T-K256R, A116T-L257G, A116T-N243V, A128S-L257G,A128S-N218S, A128S-N243V, A128S-S248N, G024E-A116T, G024E-A128S,G024E-G131H, G024E-N243V, G024E-S248N, G024E-S249A, G024E-T158S,G131H-N243V, G131H-T158S, G169A-N218S, G169A-N243V, G169A-S248N,K256R-L257G, N109G-A128S, N109G-G131H, N109G-N218S, N109G-N243V,N109G-S249A, N218S, N218S-K256R, N218S-N243V, N218S-S249A, N243V,N243V-K256R, N243V-L257G, N243V-S248N, Q103H-N109G, Q103H-N218S,S033T-A128S, S033T-L257G, S033T-N218S, S033T-S162G, S033T-S248N,S033T-T158S, S063G-K256R, S063G-L257G, S162G, S162G-G169A, S162G-S248N,S248N, S248N-K256R, S248N-L257G, S249A, T158S-S162G, and T158S-S248N,wherein amino acid positions of the variant are numbered bycorrespondence with the sequence of SEQ ID NO:2. Such variants haveenhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36,and a greater PI value than BPN′, BPN′-v3 and BPN′-v36 in this assay.The invention includes a protease variant having enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2), enhanced proteolytic activitycompared to BPN′, BPN′-v3, and BPN′-v36, a PI value of greater than 1.0to about 5 relative to BPN′-v3, and/or a PI value of greater than 1.0 toabout 5 relative to BPN′-v36 in this BMI microswatch cleaning assay, thevariant comprising an amino acid sequence having at least 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity toSEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto about 1.0 relative to BPN′-v36 in a grass microswatch cleaning assayin Detergent Composition 4 at pH 8 and 16° C.:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of BPN′-v36, A001E-A088T, A001E-A116T,A088T-A128S, A088T-N243V, A088T-Q103H, A088T-S248N, A088T-S249A, A116T,A116T-G169A, A116T-N218S, A116T-S162G, A116T-S249A, A116T-T158S,A128S-G169A, A128S-K256R, A128S-S162G, A128S-S249A, A128S-T158S,G024E-A088T, G024E-K043Y, G024E-N218S, G024E-Q103H, G024E-S063G,G024E-S162G, G131H-G169A, G131H-K256R, G131H-N218S, G131H-S162G,G131H-S248N, G131H-S249A, G169A, G169A-L257G, G169A-S249A, N076D,N076D-K256R, N076D-L257G, N076D-S162G, N076D-S249A, N109G-K256R,N109G-L257G, N109G-S248N, N243V-S249A, Q103H-A116T, Q103H-G169A,Q103H-K256R, Q103H-L257G, Q103H-N243V, Q103H-S162G, S033T-G131H,S033T-G169A, S033T-K043Y, S033T-N076D, S033T-Q206D, S063G, S063G-A116T,S063G-A128S, S063G-N243V, S063G-S162G, S063G-S248N, S063G-S249A,S063G-T158S, S249A-L257G, T158S-G169A, and T158S-K256R, wherein aminoacid positions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. Such variants have enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′in this assay. The invention includes a protease variant having enhancedproteolytic activity compared to BPN′ (SEQ ID NO:2), a PI value of 1.0relative to BPN′-v3, and a PI value of 1.0 relative to BPN′-v36 in thisBMI microswatch cleaning assay, the variant comprising an amino acidsequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 andcomprising at least one set of amino acid substitutions selected fromsaid group above, wherein amino acid positions of the variant arenumbered by correspondence with amino acid positions of the SEQ ID NO:2sequence. Also included are compositions, including, but not limited to,e.g., cleaning compositions, comprising at least one such variant andmethods for cleaning utilizing at least one such variant as described ingreater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto or greater than 0.5 and less than 0.9 relative to BPN′-v36 in a grassmicroswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of A001E-G169A, A001E-K256R, A001E-N109G,A001E-N218S, A088T-A116T, A088T-G131H, A088T-N109G, A088T-N218S,A116T-A128S, A116T-G131H, A116T-S248N, A128S, A128S-G131H, G024E,G024E-N109G, G131H, G131H-L257G, G169A-K256R, G169A-Q206D, K043Y,K043Y-A088T, K043Y-L257G, K043Y-N109G, N076D-A088T, N076D-G131H,N076D-G169A, N076D-N243V, N076D-T158S, N109G, N109G-S162G, N109G-T158S,N218S-S248N, Q103H-A128S, Q103H-S248N, Q103H-S249A, Q103H-T158S,Q206D-K256R, Q206D-L257G, Q206D-N218S, Q206D-N243V, Q206D-S248N, S033T,S033T-K256R, S033T-S063G, S033T-S249A, S063G-A088T, S063G-G131H,S063G-G169A, S063G-N109G, S162G-Q206D, S162G-S249A, S248N-S249A,S249A-K256R, T158S-Q206D, T158S-S249A, A001E-L257G, A001E-N243V,A001E-Q103H, A001E-S063G, A001E-S162G, A001E-T158S, G024E-N076D,G131H-Q206D, K043Y-A116T, K043Y-G169A, K043Y-K256R, K043Y-N076D,K043Y-S063G, K043Y-S162G, K043Y-S248N, K043Y-S249A, K043Y-T158S,N076D-A116T, N076D-A128S, N076D-S248N, N109G-G169A, Q103H, Q103H-G131H,Q206D-S249A, S063G-N076D, S063G-N218S, S063G-Q103H, A001E-A128S,A001E-G024E, A001E-G131H, A001E-N076D, A001E-Q206D, A001E-S033T,A001E-S248N, A001E-S249A, A088T-Q206D, A116T-Q206D, A128S-Q206D,G024E-Q206D, K043Y-A128S, K043Y-G131H, K043Y-N218S, K043Y-Q103H,N076D-N109G, N076D-N218S, N076D-Q103H, N109G-Q206D, Q103H-Q206D, Q206D,A001E, A001E-K043Y, K043Y-N243V, S063G-Q206D, N076D-Q206D, wherein aminoacid positions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. Such variants have proteolytic activity. Theinvention includes a protease variant having proteolytic activity and/ora PI value equal to or greater than 0.5 and less than 0.9 relative toBPN′-v36 in this grass microswatch cleaning assay, the variantcomprising an amino acid sequence having at least 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ IDNO:2 or SEQ ID NO:6 and comprising at least one set of acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI valuegreater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from greater than 1.0 to about 10, from greater than1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36in an AAPF proteolytic assay: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Qamino acid sequence (SEQ ID NO:6) comprising at least one set of aminoacid substitutions selected from the group consisting ofS033T-N076D-A128S-N218S, A001E-S033T-N109G-N218S, S033T-N218S,S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243V, A128S-G169A,S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243P,S018T-Y021N-S033T-N109G-A128S-N243V-S248N-K256R,S033T-A128S-G131H-N243P, P040E-N109G-A128S-G131H, S033T-A128S,S033T-N109G-A128S-N243V-S248N-K256R, N109G-G169A,S063G-N109G-A128S-G131H, G169A, N109G-A128S-G131H-N243V-S248N-K256R,S033T-A128S-G131H-N243V, A128S-N218S, A001E-G169A, A088T-G169A,G169A-L257G, N109G-N218S, S033T-N109G-A128S-N243P-S248N-K256R,G169A-K256R, N076D-G169A, A001E-G131H-G169A-N243V, G169A-S249A,S033T-N109G, G169A-S248N, K043Y-G169A, K043Y-N218S, N218S-L257G,N218S-N243V, S063G-G169A, A001E-A128S-G131H-N243V,A001E-S033T-N109G-N243V, A088T-N218S, G024E-N218S, G024E-S033T,G169A-Q206D, N076D-N218S, S033T-L257G, S162G-G169A, A001E-N218S,A116T-N218S, G169A-N243V, N218S, P040A-N109G-A128S-N243V-S248N-K256R,S033T-N076D, A001E-S033T, A128S-G131H, N218S-S248N,S018T-Y021N-N109G-A128S, S033T-K043Y, S033T-N243V, S033T-Q206D,S063G-N218S, S162G-N218S, T158S-G169A, A116T-G169A, G131H-G169A,N061S-N109G-A128S-S260P, N109G-A128S-N243V-K256R,N109G-A128S-N243V-S248A, N109G-A128S-N243V-S248A-K256R,N109G-A128S-N243V-S248N-K256R-L257G, N218S-K256R,S009T-N109G-A128S-K141R, S009T-S018T-Y021N-N109G-A128S-K141R,S033T-A088T, S033T-S063G, S033T-S162G, T158S-N218S,A001E-N076D-N109G-A128S, N109G-A128S-N243V-S248N-K256R,N109G-A128S-S248N-K256R, S009T-N109G-A128S-K141R-N243V,S018T-Y021N-N061S-N109G-A128S-S260P, S033T-A116T, S033T-S248N,S033T-S249A, S033T-T158S, G131H-N218S, N109A-A128S-N243V-K256R,N109G-A128S, N109G-A128S-S162G-N243V-S248N-K256R,N109G-A128S-T158S-N243V-S248N-K256R, N218S-S249A, Q206D-N218S,S018T-Y021N-N109G-A128S-N243V,S018T-Y021N-N109G-A128S-N243V-S248N-K256R, S033T-K256R, A116T-A128S,N061S-N109G-A128S-N243V-S260P, N109G-A128S-N243V-S248N,S009T-N109G-A128S-K141R-N243V-S248N-K256R, G024E-A128S,N061S-N109G-A128S-N243V-S248N-K256R-S260P, N109S-A128S-N243V-K256R,S033T, S033T-G131H, A001E-A128S, A128S, A128S-L257G, A128S-Q206D,N109Q-A128S-N243V-K256R, S009T-A128S-K141R-N243V,S009T-S018T-Y021N-A128S-K141R-N243V, A088T-A128S, A128S-K256R,A128S-N243V, N061P-N109G-N243V, N061S-A128S-N243V-S260P,S018T-Y021N-A128S-N243V, A128S-N243V-S248N-K256R, A128S-S248N,A128S-S249A, N076D-A128S, S063G-A128S, A128S-S162G, A128S-T158S,S018T-Y021N-N061S-A128S-N243V-S260P, S033T-Q103H-A128S-G131H,N061S-N109G-N243V, K043Y-A128S, N061P-N109G-G131H-N243V, N109G-L257G,A001E-G024E-S204E-Q206D, A001E-L257G, A088T-N109G, G024E-N109G,K043Y-N109G, N061G-N109G-N243V, N076D-N109G, N109G, N109G-A116T,N109G-K256R, N109G-N243V-K256R, N109G-N243V-S248A-K256R, N109G-Q206D,S063G-N109G, A001E-A116T, A001E-N109G, A001E-Q206D, A088T-A116T,A088T-N243V, A116T-L257G, G024E-A116T, G024E-L257G, G024E-N243V,G024E-Q206D, N109G-G131H, N109G-N243V, N109G-S162G, N109G-S248N,N109G-S248N-K256R, N109G-S249A, N109G-T158S, N243V-L257G, A001E-A088T,A001E-G024E, A001E-K256R, A001E-N076D, A001E-N243V, A088T, A088T-L257G,A088T-Q206D, A116T, A116T-K256R, A116T-N243V, G024E-A088T, G024E-K043Y,G024E-K256R, G024E-N076D, G024E-S162G, G024E-S248N, K043Y-A088T,K043Y-A116T, K043Y-L257G, K043Y-N243V, K043Y-Q206D, K256R-L257G,N076D-A116T, N076D-L257G, N076D-N243V, N076D-Q206D, N109G-N243V-S248N,N109G-N243V-S248N-K256R, N243V-K256R, Q206D, Q206D-L257G, Q206D-N243V,Q206D-S248N, S063G-K256R, S063G-L257G, T158S-L257G, A001E, A001E-K043Y,A001E-S162G, A001E-S248N, A001E-S249A, A001E-T158S, A088T-K256R,A088T-S162G, A088T-S248N, A088T-S249A, A116T-Q206D, A116T-S248N,A116T-S249A, G024E, G024E-G131H, G024E-S249A, G024E-T158S, G131H,G131H-K256R, G131H-L257G, K043Y-K256R, K043Y-N076D, K256R, L257G,N076D-A088T, N076D-K256R, N076D-S162G, N076D-S248N, N076D-S249A,N109G-N243P-S248A-K256R, N109G-N243P-S248N-K256R, N243V, Q206D-K256R,S033T-P040E-Q103H-N109G, S063G, S063G-A116T, S063G-Q206D, S162G-K256R,S162G-L257G, S162G-N243V, S162G-Q206D, S162G-S248N, S248N, S248N-L257G,S249A, S249A-L257G, T158S, T158S-N243V, and T158S-Q206D, wherein aminoacid positions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. Such variants have enhanced proteolyticactivity compared to BPN′, BPN′-v3, and BPN′-v36, and a greater PI valuethan BPN′, BPN′-v3 and BPN′-v36 in this assay. The invention includes aprotease variant having enhanced proteolytic activity compared to BPN′(SEQ ID NO:2), enhanced proteolytic activity compared to BPN′, BPN′-v3,and BPN′-v36, a PI value of greater than 1.0 to about 5 relative toBPN′-v3, and/or a PI value of greater than 1.0 to about 5 relative toBPN′-v36 in this assay, the variant comprising an amino acid sequencehaving at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprisingat least one set of amino acid substitutions selected from said groupabove, wherein amino acid positions of the variant are numbered bycorrespondence with amino acid positions of the SEQ ID NO:2 sequence.Also included are compositions, including, but not limited to, e.g.,cleaning compositions, comprising at least one such variant and methodsfor cleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto about 1.0 relative to BPN′-v36 in an AAPF proteolytic assay:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of BPN′-v36, A001E-G131H, A001E-S063G,A088T-G131H, A088T-T158S, A116T-G131H, A116T-S162G, A116T-T158S,G024E-S063G,

G131H-N243V, G131H-N243V-K256R, G131H-Q206D, G131H-S249A, K043Y,K043Y-S063G, K043Y-S248N, K043Y-S249A, K043Y-T158S, N076D, N076D-G131H,N076D-T158S, N243V-S248N, N243V-S248N-K256R, N243V-S249A, Q103H-G169A,Q206D-S249A, S063G-N076D, S063G-N243V, S063G-S162G, S063G-S249A,S063G-T158S, S162G, S162G-S249A, S248N-K256R, S248N-S249A, S249A-K256R,T158S-K256R, T158S-S248N, and T158S-S249A, wherein amino acid positionsof the variant are numbered by correspondence with the sequence of SEQID NO:2. Such variants have enhanced proteolytic activity compared toBPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. Theinvention includes a protease variant having enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2), a PI value of 1.0 relative toBPN′-v3, and a PI value of 1.0 relative to BPN′-v36 in this assay, thevariant comprising an amino acid sequence having at least 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity toSEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto about 0.9 relative to BPN′-v36 in an AAPF proteolytic assay:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of G131H-S162G, G131H-S248N, G131H-T158S,K043Y-G131H, K043Y-S162G, S063G-A088T, S063G-G131H, S063G-S248N,T158S-S162G, Q103H-N218S, S033T-Q103H, and Q103H-A128S, wherein aminoacid positions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. Such variants have proteolytic activity. Theinvention includes a protease variant having proteolytic activity and/ora PI value of 0.9 relative to BPN′-v36 in this assay, the variantcomprising an amino acid sequence having at least 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ IDNO:2 or SEQ ID NO:6 and comprising at least one set of acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

Also provided is a subtilisin protease variant having proteolyticactivity, enhanced proteolytic activity compared to BPN′, or a PI valuegreater than that of BPN′ (SEQ ID NO:2) in a BMI microswatch cleaningassay, the variant comprising an amino acid sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2,wherein the variant comprises at least one substitution selected fromthe group of X001E, X009T, X018T, X021N, X024G, X033T, X040A, X043Y,X061G/P/S, X063G, X076D, X088T, X103H, X109A/G/Q/S, X116T, X128S, X131H,X141R, X158S, X162G, X169A, X204E, X206D, X218S, X243P/V, X248A/N,X249A, X256R, X257G, and X260P, wherein positions of the variant arenumbered by correspondence with the sequence of SEQ ID NO:2, andoptionally wherein the variant comprises at least one substitutionselected from the group of A001E, S009T, S018T, Y021N, S024G, S033T,P040A, K043Y, N061G/P/S, S063G, N076D, A088T, Q103H, N109A/G/Q/S, A116T,G128S, G131H, K141R, T158S, S162G, G169A, S204E, Q206D, N218S, N243P/V,S248A/N, S249A, K256R, L257G, and S260P. Also included are compositions,including cleaning compositions, comprising at least one such proteasevariant and methods for cleaning utilizing at least one such proteasevariant as described in greater detail elsewhere herein.

Example 9 Construction and Cleaning Performance of Variants from aCombinatorial Library Based on BPN′-v36 Parent

A BPN′ combinatorial library based on the BPN′-v36 parent molecule wasmade by DNA 2.0 and delivered as a ligation reaction. For efficienttransformation of B. subtilis, DNA from the ligation reaction mixturewas amplified before transformation and transformants grown as describedin Example 2. The variants were tested for cleaning performance usingBMI microswatch assay in Detergent Composition 4 at 16° C. and pH 8 andegg microswatch assay in Detergent Composition 4 at 16° C. and pH 8.Protein content was determined using the TCA assay. Assays wereperformed as in Example 1 and Performance Indices were calculatedrelative to BPN′-v36 (i.e., BPN′-S24G-S53G-S78N-S101N-G128A-Y217Q).

The following BPN′-v36 variants were determined to have a PI valuegreater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from greater than 1.0 to about 10, from greater than1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence(SEQ ID NO:6) comprising at least one set of amino acid substitutionsselected from the group consisting of A088T-A116T-N243V-K256R-L257G,A088T-A116T-N243V-L257G, A088T-T158S-N218S-K256R,A088T-T158S-N218S-N243V-L257G,A088T-A116T-T158S-N218S-N243V-K256R-L257G,A088T-N109G-A116T-G131H-A153S-N218S-S248N-L257G,A088T-N109G-A116T-T158S-S248N-K256R-L257G, A088T-N109G-T158S-L257G,A114S-A116T-N218S-N243V-S248N-K256R-L257G, A116T-T158S-K256R,A088T-A116T-G131H-T158S-S248N-L257G, A088T-A116T-T158S,A088T-N109G-A116T-G131H-L257G,A088T-N109G-A116T-T158S-N243V-S248N-L257G, A088T-N109G-N243V-L257G,A088T-N109G-N243V-S248N, A088T-N109G-T158S-N243V-L257G,A088T-N109G-T158S-N243V-S248N-L257G, A116T-T158S-S248N-L257G,Y006H-A116T-G131H-S248N, A088T-A116T-G131H-T158S-N218S-N243V,A088T-A116T-G131H-T158S-N243V,A088T-A116T-G131H-T158S-N243V-K256R-L257G,A088T-A116T-N218S-N243V-K256R-L257G, A088T-A116T-S248N-K256R-L257G,A088T-A116T-T158S-N218S-N243V, A088T-A116T-T158S-N243V-K256R-L257G,A088T-A116T-T158S-N243V-S248N-L257G,A088T-G131H-N243V-S248N-K256R-L257G, A088T-N109G-A116T-T158S-L257G,A088T-N109G-A116T-T158S-N212D-N243V-K256R-L257G,A088T-N109G-A116T-T158S-N218S-N243V-S248N-K256R,A088T-N109G-A116T-T158S-S248N-L257G,A088T-N109G-G131H-V148A-N218S-N243V-K256R-L257G, A088T-N109G-K256R,A088T-N109G-N243V-S248N-L257G, A088T-N109G-T158S-K256R,A088T-N109G-T158S-N243V, A088T-T158S-N243V-K256R-L257G, A116T,A116T-N218S-N243V-L257G-N269S, A116T-T158S-K256R-L257G,N109G-A116T-K256R-L257G, N109G-A116T-N243V,N109G-A116T-T158S-N243V-K256R-L257G, N109G-G131H-L257G,N109G-G131H-S248N-K256R-L257G, N109G-G131H-T158S-K256R-L257G,S003P-A116T-T158S-S248N-K256R, T158S-S248N-K256R,A088T-A116T-G131H-N243V-K256R, A088T-A116T-G131H-S248N-K256R-L257G,A088T-A116T-G131H-V147A-T158S-N218S-N243V-S248N-L257G,A088T-A116T-S248N-L257G, A088T-A116T-T158S-N218S,A088T-A116T-T158S-N218S-K256R-L257G, A088T-A116T-T158S-N218S-L257G,A088T-G131H-N243V-L257G, A088T-G131H-T158S-S248N-L257G, A088T-L257G,A088T-N109G-A116T, A088T-N109G-A116T-G131H-N218S,A088T-N109G-A116T-G131H-N218S-S248N-L257G,A088T-N109G-A116T-G131H-N243V-S248N-K256R-L257G,A088T-N109G-A116T-G131H-T158S-S248N-K256R-L257G,A088T-N109G-A116T-N218S-N243V-K256R,A088T-N109G-A116T-N218S-N243V-L257G,A088T-N109G-A116T-N243V-S248N-K256R,A088T-N109G-A116T-N243V-S248N-K256R-L257G,A088T-N109G-A116T-T158S-L257G, A088T-N109G-A116T-T158S-N243V-L257G,A088T-N109G-G131H-T158S-N243V-S248N-K256R,A088T-N109G-G131H-T158S-W241R-S248N-K256R, A088T-N109G-K256R-L257G,A088T-N109G-L257G, A088T-N109G-N243V, A088T-N109G-N243V-K256R,A088T-N109G-N243V-K256R-L257G, A088T-N109G-S248N-K256R,A088T-N109G-T158S-N218S-K256R-L257G,A088T-N109G-T158S-N218S-N243V-S248N-K256R,A088T-N109G-T158S-N243V-K256R, A088T-N109G-T158S-N243V-K256R-L257G,A088T-N109G-T158S-N243V-S248N-A274D, A088T-N109G-T158S-S248N-L257G,A088T-T158S-K256R, A088T-T158S-N218S-N243V-K256R-L257G,A088T-T158S-N243V-L257G, A116T-G131H-N218S-N243V-S248N,A116T-G131H-S248N-L257G, A116T-S248N-K256R-L257G,A116T-T158S-N218S-N243V-K256R, A116T-T158S-N218S-S248N-L257G-Q271R,A116T-T158S-N243V-K256R-L257G, A116T-T158S-N243V-S248N-L257G,G131H-S248N, G131H-T158S-I234T-N243V-K256R,G131H-W241L-N243V-S248N-K256R,N109G-A116T-G131H-A137V-T158S-S248N-K256R-L257G,N109G-A116T-G131H-A151S-N218S-K256R-L257G,N109G-A116T-G131H-T158S-N218S-N243V-K256R,N109G-A116T-G131H-T158S-N218S-S248N,N109G-A116T-G131H-T158S-N243V-S248N, N109G-A116T-S248N,N109G-A116T-T158S-L257G, N109G-A116T-T158S-N218S-W241R-N243V,N109G-A116T-T158S-N243V-S248N-L257G,N109G-A116T-T158S-S248N-K256R-L257G, N109G-A116T-T158S-S248N-L257G,N109G-G131H-N218S-L257G, N109G-G131H-N218S-S248N-K256R-L257G,N109G-G131H-T158S-N218S-S248N-K256R-L257G-A274T, N109G-K256R,N109G-N243V-L257G, N109G-T158S-N218S-K256R-L257G,N109G-T158S-N218S-L257G, N109G-T158S-S248N-K256R,P014L-A015L-L016C-H017T-S018L-Q019K-G020A-Y021T-T022L-G023E,S003F-A088T-N109G-A116T-T158S-N243V-K256R-L257G,V004A-A088T-A116T-T158S-N218S,V004A-N109G-A116T-G131H-S248N-K256R-L257G,V004L-A116T-N218S-N243V-S248N-L257G, Y006H-N109G-N218S-N243V-S248N,A001T-A116T-T158S-N243V-L257G, A088T-A116T, A088T-A116T-G131H-L257G,A088T-A116T-G131H-N218S-L257G,A088T-A116T-G131H-N218S-S248N-K256R-L257G,A088T-A116T-G131H-N218S-S248N-L257G,A088T-A116T-G131H-N243V-K256R-L257G, A088T-A116T-G131H-N243V-L257G,A088T-A116T-G131H-N243V-S248N, A088T-A116T-G131H-T158S-K256R-L257G,A088T-A116T-G131H-T158S-L257G, A088T-A116T-G131H-T158S-N218S,A088T-A116T-G131H-T158S-N218S-N243V-K256R-A273T,A088T-A116T-G131H-T158S-N218S-S248N-K256R,A088T-A116T-G131H-T158S-N218S-S248N-L257G,A088T-A116T-G131H-T158S-N243V-S248N-K256R,A088T-A116T-G131H-T158S-S248N, A088T-A116T-G131H-T158S-S248N-L257G,A088T-A116T-K256R, A088T-A116T-K256R-L257G,A088T-A116T-N218S-N243V-L257G, A088T-A116T-N243V-K256R,A088T-A116T-N243V-L257G, A088T-A116T-N243V-S248N-K256R-L257G,A088T-A116T-S248N-K256R, A088T-A116T-T158S-K256R,A088T-A116T-T158S-N218S, A088T-A116T-T158S-N218S-N243V-K256R,A088T-A116T-T158S-N218S-N243V-K256R-N269S,A088T-A116T-T158S-N218S-N243V-S248N,A088T-A116T-T158S-N218S-N243V-S248N,A088T-A116T-T158S-N218S-N243V-S248N-K256R-L257G,A088T-A116T-T158S-N243V-K256R, A088T-A116T-T158S-N243V-L257G,A088T-A116T-T158S-N243V-S248N-K256R,A088T-A116T-T158S-N243V-S248N-K256R-L257G,A088T-A116T-T158S-S248N-K256R, A088T-A116T-V143A-N218S-S248N-K256R,A088T-A116T-V147I-T158S-N218S-N243V-L257G, A088T-G131H-K256R-L257G,A088T-G131H-N218S-N243V-S248N, A088T-G131H-N218S-S248N-L257G,A088T-G131H-S248N-K256R-L257G, A088T-G131H-T158S-L257G,A088T-G131H-T158S-N218S-K256R,A088T-G131H-T158S-N218S-N243V-K256R-L257G,A088T-G131H-T158S-N218S-N243V-L257G, A088T-G131H-T158S-N218S-S248N,A088T-G131H-T158S-N243V, A088T-G131H-T158S-N243V,A088T-G131H-T158S-N243V-S248N, A088T-G131H-T158S-N243V-S248N-K256R,A088T-G131H-T158S-N243V-S248N-L257G,A088T-I107T-N109G-A116T-G131H-T158S-N218S-N243V-S248N,A088T-N109G-A116T-G131H-L257G, A088T-N109G-A116T-G131H-N218S,A088T-N109G-A116T-G131H-N218S-L257G,A088T-N109G-A116T-G131H-N218S-N243V,A088T-N109G-A116T-G131H-N218S-N243V-K256R-L257G,A088T-N109G-A116T-G131H-N218S-N243V-L257G,A088T-N109G-A116T-G131H-N218S-S248N-K256R-L257G,A088T-N109G-A116T-G131H-N243V, A088T-N109G-A116T-G131H-N243V-L257G,A088T-N109G-A116T-G131H-N243V-S248N-L257G,A088T-N109G-A116T-G131H-S248N, A088T-N109G-A116T-G131H-S248N-K256R,A088T-N109G-A116T-G131H-S248N-L257G,A088T-N109G-A116T-G131H-T158S-L257G,A088T-N109G-A116T-G131H-T158S-N218S,A088T-N109G-A116T-G131H-T158S-N218S-S248N-K256R,A088T-N109G-A116T-G131H-T158S-N218T-N243V,A088T-N109G-A116T-G131H-T158S-N243V-K256R,A088T-N109G-A116T-G131H-T158S-N243V-K256R-L257G,A088T-N109G-A116T-G131H-T158S-N243V-S248N,A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R,A088T-N109G-A116T-G131H-T158S-S248N-K256R-L257G,A088T-N109G-A116T-G131H-T158S-S248N-L257G, A088T-N109G-A116T-N218S,A088T-N109G-A116T-N218S-L257G, A088T-N109G-A116T-N218S-N243V,A088T-N109G-A116T-N218S-N243V-S248N-L257G,A088T-N109G-A116T-N218S-S248N-K256R, A088T-N109G-A116T-N218T-K256R,A088T-N109G-K256R, A088T-N109G-A116T-N218T-K256R,A088T-N109G-A116T-N218T-K256R-L257G, A088T-N109G-A116T-N243V,A088T-N109G-A116T-N243V-K256R-L257G,A088T-N109G-A116T-N243V-K256R-L257G-N269D,A088T-N109G-A116T-S248N-K256R, A088T-N109G-A116T-T158S,A088T-N109G-A116T-T158S-N218S-L257G,A088T-N109G-A116T-T158S-N218S-N243V,A088T-N109G-A116T-T158S-N218S-N243V-K256R,A088T-N109G-A116T-T158S-N218S-N243V-K256R-L257G,A088T-N109G-A116T-T158S-N218S-N243V-K256R-L257G,A088T-N109G-A116T-T158S-N218S-N243V-L257G,A088T-N109G-A116T-T158S-N218S-S248N, A088T-N109G-A116T-T158S-N243V,A088T-N109G-A116T-T158S-N243V-K256R,A088T-N109G-A116T-T158S-N243V-K256R-L257G,A088T-N109G-A116T-T158S-S248N-L257G, A088T-N109G-G131H-L257G,A088T-N109G-G131H-N218S-K256R-L257G,A088T-N109G-G131H-N218S-N243V-K256R,A088T-N109G-G131H-N218S-N243V-L257G,A088T-N109G-G131H-N218S-N243V-S248N-K256R-L257G,A088T-N109G-G131H-N243V, A088T-N109G-G131H-N243V-L257G,A088T-N109G-G131H-N243V-S248N-K256R,A088T-N109G-G131H-N243V-S248N-L257G, A088T-N109G-G131H-S248N-L257G,A088T-N109G-G131H-T158S-L257G,A088T-N109G-G131H-T158S-N218S-N243V-S248N-K256R,A088T-N109G-G131H-T158S-N243V, A088T-N109G-G131H-T158S-N243V-K256R,A088T-N109G-G131H-T158S-N243V-K256R-L257G,A088T-N109G-G131H-T158S-N243V-L257G, A088T-N109G-L257G,A088T-N109G-N218S-K256R, A088T-N109G-N218S-N243V-S248N-L257G,A088T-N109G-N218S-S248N-K256R-L257G, A088T-N109G-N243V-K256R-L257G,A088T-N109G-N243V-S248N-K256R-L257G,A088T-N109G-N243V-S248N-L257G-I268V, A088T-N109G-S248N-K256R-L257G,A088T-N109G-T158S-N218S-K256R, A088T-N109G-T158S-N218S-N243V-L257G,A088T-N109G-T158S-N218S-N243V-L257G,A088T-N109G-T158S-N243V-K256R-I268V,A088T-N109G-T158S-N243V-S248N-Q275R, A088T-N218S-N243V,A088T-N218S-N243V-S248N-K256R-L257G, A088T-N218S-S248N,A088T-N218S-S248N-L257G, A088T-N243V, A088T-N243V, A088T-N243V-K256R,A088T-N243V-L257G, A088T-S145T-T158S-S248N, A088T-T158S-L257G,A088T-T158S-N218S-S248N-L257G, A088T-T158S-N243V-K256R-L257G-Q271H,A088T-T158S-S248N, A088T-V143A-T158S-K256R, A116T-G131H-K256R,A116T-G131H-N218S, A116T-G131H-N243V, A116T-G131H-N243V-K256R,A116T-G131H-N243V-L257G, A116T-G131H-S248N-K256R,A116T-G131H-T158S-N218S-I234T-N243V-S248N-K256R,A116T-G131H-T158S-N243V-L257G, A116T-G131H-T158S-N243V-S248N-K256R,A116T-G131H-V143F-T158S-N218S, A116T-L257G, A116T-N218S,A116T-N218S-L257G, A116T-N218S-N243V-L257G, A116T-N243V,A116T-N243V-K256R, A116T-N243V-S248N, A116T-N243V-S248N-K256R-L257G,A116T-S248N, A116T-T158S, A116T-T158S-N218S-N243V,A116T-T158S-N218S-S248N, A116T-T158S-N243V, A116T-T158S-N243V-K256R,A116T-T158S-N243V-L257G, A116T-T158S-N243V-S248N,A116T-T158S-S248N-K256R-L257G,A116T-V149I-T158S-N243V-S248N-K256R-Q271H, G131H-N218S-N243V-L257G,G131H-N243V, G131H-N243V-S248N-K256R, G131H-T158S,G131H-T158S-N218S-N243V-K256R, G131H-T158S-N243V-K256R-L257G,G131H-T158S-N243V-S248N-L257G, N109G-A116T-G131H-N218S-K256R-L257G,N109G-A116T-G131H-N218S-L257G,N109G-A116T-G131H-N218S-N243V-K256R-L257G,N109G-A116T-G131H-N218S-S248N-K256R, N109G-A116T-G131H-N243V-K256R,N109G-A116T-G131H-N243V-L257G,N109G-A116T-G131H-N243V-S248N-K256R-L257G, N109G-A116T-G131H-S248N,N109G-A116T-G131H-S248N-I268V,N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R,N109G-A116T-G131H-T158S-N218S-S248N-L257G,N109G-A116T-G131H-T158S-S248N, N109G-A116T-G131H-T158S-S248N-K256R,N109G-A116T-N218S, N109G-A116T-N218S-N243V-K256R,N109G-A116T-N218S-N243V-K256R-L257G, N109G-A116T-N218S-S248N-L257G,N109G-A116T-N243V-K256R, N109G-A116T-N243V-S248N-K256R-L257G,N109G-A116T-S248N-L257G, N109G-A116T-T158S-G211V-N243V-S248N-K256R,N109G-A116T-T158S-K256R-L257G, N109G-A116T-T158S-N218S,N109G-A116T-T158S-N218S-N243V-K256R-L257G,N109G-A116T-T158S-N218S-N243V-L257G,N109G-A116T-T158S-N218S-N243V-S248N-L257G,N109G-A116T-T158S-N218S-S248N-K256R-L257G, N109G-A116T-T158S-N243V,N109G-A116T-T158S-Q275R, N109G-G131H-A137V-T158S-N218S-S248N,N109G-G131H-N218S-K237N, N109G-G131H-N218S-N243V-K256R-L257G,N109G-G131H-N218S-S248N-K256R, N109G-G131H-N243V-K256R-L257G,N109G-G131H-S145F-N218S-N243V-K256R-L257G, N109G-G131H-S248N-K256R,N109G-G131H-S248N-L257G, N109G-G131H-T158S-K256R,N109G-G131H-T158S-N218S-N243V-K256R, N109G-G131H-T158S-N243V,N109G-G131H-T158S-N243V-K256R-L257G, N109G-G131H-T158S-N243V-L257G,N109G-G131H-T158S-S248N-L257G, N109G-G131H-T158S-S248N-Q271R,N109G-N218S-L257G, N109G-N218S-N243V, N109G-N243V-K256R-L257G,N109G-N243V-S248N-K256R-L257G, N109G-T158S-I268V, N109G-T158S-K256R,N109G-T158S-N218S-N243V-K256R-L257G, N109G-T158S-N218S-S248N-L257G,N109G-T158S-N243V, N109G-T158S-N243V-K256R-L257G,N109G-T158S-N243V-S248N, N109S-A116T-S248N, N218S,N218S-N243V-S248N-K256R-L257G, N218S-S248N-L257G, N243V, N243V-K256R,N243V-S248N-K256R, N243V-S248N-K256R-L257G,S105P-A116T-T158S-N218S-N243V-S248N-K256R, S248N, T158S-N243V-K256R, andT158S-N243V-L257G, wherein amino acid positions of the variant arenumbered by correspondence with the sequence of SEQ ID NO:2. Suchvariants have enhanced proteolytic activity compared to BPN′, BPN′-v3,and BPN′-v36, and a greater PI value than BPN′, BPN′-v3 and BPN′-v36 inthis assay. The invention includes a protease variant having enhancedproteolytic activity compared to BPN′ (SEQ ID NO:2), enhancedproteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, a PI valueof greater than 1.0 to about 5 relative to BPN′-v3, and/or a PI value ofgreater than 1.0 to about 5 relative to BPN′-v36 in this assay, thevariant comprising an amino acid sequence having at least 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity toSEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto about 1.0 relative to BPN′-v36 in a BMI microswatch cleaning assay(Test Method 3) in Detergent Composition 4 at pH 8 and 16° C.:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of BPN′-v36, A088T, A088T-A116T-G131H-L257G,A088T-A116T-G131H-N218S-A274T, A088T-A116T-G131H-N218S-K256R,A088T-A116T-G131H-N218S-K256R-L257G, A088T-A116T-G131H-N218S-N243V,A088T-A116T-G131H-N218S-N243V-L257G,A088T-A116T-G131H-N218S-N243V-S248N-K256R,A088T-A116T-G131H-N218S-N243V-S248N-K256R-L257G,A088T-A116T-G131H-N218S-N243V-S248N-L257G,A088T-A116T-G131H-N218S-S248N, A088T-A116T-G131H-N218S-S248N-L257G,A088T-A116T-G131H-N243V, A088T-A116T-G131H-N243V-K256R,A088T-A116T-G131H-N243V-S248N, A088T-A116T-G131H-N243V-S248N-A274V,A088T-A116T-G131H-N243V-S248N-K256R-L257G,A088T-A116T-G131H-N243V-S248N-K256R-L257G,A088T-A116T-G131H-N243V-S248N-L257G, A088T-A116T-G131H-S248N-K256R,A088T-A116T-G131H-S248N-K256R-L257G, A088T-A116T-G131H-T158S-K256R,A088T-A116T-G131H-T158S-K256R-L257G,A088T-A116T-G131H-T158S-N218S-K256R,A088T-A116T-G131H-T158S-N218S-K256R-L257G,A088T-A116T-G131H-T158S-N218S-L257G,A088T-A116T-G131H-T158S-N218S-N243V-K256R,A088T-A116T-G131H-T158S-N218S-N243V-K256R-L257G,A088T-A116T-G131H-T158S-N218S-N243V-L257G,A088T-A116T-G131H-T158S-N218S-N243V-S248N,A088T-A116T-G131H-T158S-N218S-N243V-S248N,A088T-A116T-G131H-T158S-N218S-N243V-S248N-K256R,A088T-A116T-G131H-T158S-N243V-K256R,A088T-A116T-G131H-T158S-N243V-K256R,A088T-A116T-G131H-T158S-N243V-K256R-L257G,A088T-A116T-G131H-T158S-N243V-L257G,A088T-A116T-G131H-T158S-N243V-S248N,A088T-A116T-G131H-T158S-N243V-S248N-K256R-L257G,A088T-A116T-G131H-T158S-N243V-S248N-K256R-L257G,A088T-A116T-G131H-T158S-N243V-S248N-L257G,A088T-A116T-G131H-T158S-S248N-K256R,A088T-A116T-G131H-T158S-S248N-K256R-L257G, A088T-A116T-K256R,A088T-A116T-L257G, A088T-A116T-N218S, A088T-A116T-N218S-N243V-K256R,A088T-A116T-N218S-N243V-N269D, A088T-A116T-N218S-N243V-S248N,A088T-A116T-N218S-N243V-S248N-K256R-L257G,A088T-A116T-N218S-N243V-S248N-L257G, A088T-A116T-N218S-S248N,A088T-A116T-N218S-S248N-L257G, A088T-A116T-N243V-K256R-L257G,A088T-A116T-N243V-S248N-K256R, A088T-A116T-S248N,A088T-A116T-S248N-K256R,A088T-A116T-T158S-A216S-N218S-N243V-K256R-L257G,A088T-A116T-T158S-N218S-K256R, A088T-A116T-T158S-N218S-N243V-K256R,A088T-A116T-T158S-N218S-N243V-S248N-K256R-L257G,A088T-A116T-T158S-N218S-S248N, A088T-A116T-T158S-N243V-K256R,A088T-A116T-T158S-N243V-S248N, A088T-A116T-T158S-N243V-S248N-K256R,A088T-A116T-T158S-N243V-S248N-L257G, A088T-A116T-T158S-S248N,A088T-G131D-T158S-N243V-S248N, A088T-G131H-A138V-N218S-L257G,A088T-G131H-K256R, A088T-G131H-N218S-N243V-K256R,A088T-G131H-N218S-N243V-K256R, A088T-G131H-N218S-S248N,A088T-G131H-N218S-S248N-K256R-L257G, A088T-G131H-N218T-L257G,A088T-G131H-N243V-L257G, A088T-G131H-N243V-S248N-K256R,A088T-G131H-S248N, A088T-G131H-S248N-L257G, A088T-G131H-T158S-K256R,A088T-G131H-T158S-K256R-L257G, A088T-G131H-T158S-N218S,A088T-G131H-T158S-N218S, A088T-G131H-T158S-N218S-K256R-L257G,A088T-G131H-T158S-N218S-N243V-K256R,A088T-G131H-T158S-N218S-N243V-K256R,A088T-G131H-T158S-N218S-N243V-S248N, A088T-G131H-T158S-N218S-S248N,A088T-G131H-T158S-N218S-S248N-K256R,A088T-G131H-T158S-N218S-S248N-L257G-I268V,A088T-G131H-T158S-N243V-K256R, A088T-G131H-T158S-N243V-K256R-L257G,A088T-G131H-T158S-N243V-S248N,A088T-G131H-T158S-N243V-S248N-K256R-L257G, A088T-G131H-T158S-S248N,A088T-G131H-T158S-S248N-K256R, A088T-N109G-A116T-G131H-K256R,A088T-N109G-A116T-G131H-K256R-L257G,A088T-N109G-A116T-G131H-K256R-L257G,A088T-N109G-A116T-G131H-N218S-K256R,A088T-N109G-A116T-G131H-N218S-K256R,A088T-N109G-A116T-G131H-N218S-K256R-L257G,A088T-N109G-A116T-G131H-N218S-N243V-L257G,A088T-N109G-A116T-G131H-N218S-S248N,A088T-N109G-A116T-G131H-N218S-S248N-L257G,A088T-N109G-A116T-G131H-N243V-K256R,A088T-N109G-A116T-G131H-N243V-K256R-L257G,A088T-N109G-A116T-G131H-N243V-S248N-K256R,A088T-N109G-A116T-G131H-N243V-S248N-K256R,A088T-N109G-A116T-G131H-S248N-K256R-L257G,A088T-N109G-A116T-G131H-S248N-K256R-L257G,A088T-N109G-A116T-G131H-S248N-L257G,A088T-N109G-A116T-G131H-T158S-K256R,A088T-N109G-A116T-G131H-T158S-L257G,A088T-N109G-A116T-G131H-T158S-N218S,A088T-N109G-A116T-G131H-T158S-N218S-L257G,A088T-N109G-A116T-G131H-T158S-N218S-N243V,A088T-N109G-A116T-G131H-T158S-N218S-N243V-K256R,A088T-N109G-A116T-G131H-T158S-N218S-N243V-L257G,A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R,A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R,A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,A088T-N109G-A116T-G131H-T158S-N218S-S248N,A088T-N109G-A116T-G131H-T158S-N218T-K256R,A088T-N109G-A116T-G131H-T158S-N243V,A088T-N109G-A116T-G131H-T158S-N243V-S248N,A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R-L257G,A088T-N109G-A116T-G131H-T158S-N243V-S248N-L257G,A088T-N109G-A116T-G131H-V149A-T158S-N218S-K256R,A088T-N109G-A116T-K256R, A088T-N109G-A116T-N218S-K256R,A088T-N109G-A116T-N218S-N243V,A088T-N109G-A116T-N218S-N243V-K256R-L257G,A088T-N109G-A116T-N218S-N243V-L257G,A088T-N109G-A116T-N218S-N243V-S248N-K256R,A088T-N109G-A116T-N218S-S248N,A088T-N109G-A116T-N218S-S248N-K256R-L257G,A088T-N109G-A116T-N243V-K256R,A088T-N109G-A116T-N243V-S248N-K256R-L257G,A088T-N109G-A116T-N243V-S248N-L257G,A088T-N109G-A116T-N243V-S248N-L257G, A088T-N109G-A116T-S248N,A088T-N109G-A116T-S248N-K256R-L257G, A088T-N109G-A116T-S248N-L257G,A088T-N109G-A116T-T158S, A088T-N109G-A116T-T158S-K256R,A088T-N109G-A116T-T158S-N218S, A088T-N109G-A116T-T158S-N218S-L257G,A088T-N109G-A116T-T158S-N218S-N243V-S248N,A088T-N109G-A116T-T158S-N218S-N243V-S248N,A088T-N109G-A116T-T158S-N218S-N243V-S248N-K256R,A088T-N109G-A116T-T158S-N218S-N243V-S248N-K256R-L257G,A088T-N109G-A116T-T158S-N218S-S248N,A088T-N109G-A116T-T158S-N218S-S248N-K256R,A088T-N109G-A116T-T158S-N218S-S248N-L257G,A088T-N109G-A116T-T158S-N218S-S248N-L257G,A088T-N109G-A116T-T158S-N243V-K256R,A088T-N109G-A116T-T158S-N243V-S248N, A088T-N109G-A116T-T158S-S248N,A088T-N109G-A116T-T158S-S248N-K256R,A088T-N109G-A137E-T158S-N218S-N243V-S248N-K256R-L257G,A088T-N109G-G131H-A152S-T158S-N218S-S248N-K256R,A088T-N109G-G131H-K256R-L257G, A088T-N109G-G131H-N218S,A088T-N109G-G131H-N218S, A088T-N109G-G131H-N218S-K256R,A088T-N109G-G131H-N218S-K256R, A088T-N109G-G131H-N218S-L257G,A088T-N109G-G131H-N218S-N243V, A088T-N109G-G131H-N218S-N243V-K256R,A088T-N109G-G131H-N218S-N243V-K256R-L257G,A088T-N109G-G131H-N218S-N243V-S248N-K256R,A088T-N109G-G131H-N218S-S248N, A088T-N109G-G131H-N218S-S248N-K256R,A088T-N109G-G131H-N218S-S248N-K256R,A088T-N109G-G131H-N218S-S248N-K256R-L257G,A088T-N109G-G131H-N243V-K256R, A088T-N109G-G131H-N243V-K256R-L257G,A088T-N109G-G131H-N243V-K256R-L257G, A088T-N109G-G131H-N243V-L257G,A088T-N109G-G131H-N243V-S248N-K256R, A088T-N109G-G131H-S248N-K256R,A088T-N109G-G131H-S248N-L257G, A088T-N109G-G131H-T158S,A088T-N109G-G131H-T158S-K256R, A088T-N109G-G131H-T158S-K256R-L257G,A088T-N109G-G131H-T158S-N218S-K256R,A088T-N109G-G131H-T158S-N218S-K256R,A088T-N109G-G131H-T158S-N218S-L257G,A088T-N109G-G131H-T158S-N218S-L257G,A088T-N109G-G131H-T158S-N218S-N243V,A088T-N109G-G131H-T158S-N218S-N243V-K256R,A088T-N109G-G131H-T158S-N218S-N243V-S248N,A088T-N109G-G131H-T158S-N218S-S248N-L257G,A088T-N109G-G131H-T158S-N243V-K256R-L257G,A088T-N109G-G131H-T158S-N243V-S248N,A088T-N109G-G131H-T158S-N243V-S248N-K256R-L257G,A088T-N109G-G131H-T158S-N243V-S248N-K256R-L257G,A088T-N109G-G131H-T158S-N243V-S248N-L257G,A088T-N109G-G131H-T158S-N243V-S248N-L257G,A088T-N109G-G131H-T158S-S248N, A088T-N109G-G131H-T158S-S248N-L257G,A088T-N109G-G131H-V149A-K256R-L257G,A088T-N109G-G154A-N155P-E156T-G157L-T158M-S159E-G160E-S161L,A088T-N109G-K256R-L257G, A088T-N109G-N218S-K256R,A088T-N109G-N218S-N243V-L257G, A088T-N109G-N218S-N243V-S248N-K256R,A088T-N109G-N218S-N243V-S248N-K256R, A088T-N109G-N218S-S248N,A088T-N109G-N218S-S248N-L257G, A088T-N109G-N218S-S248N-L257G,A088T-N109G-N243V-S248N-K256R, A088T-N109G-S248N, A088T-N109G-S248N,A088T-N109G-T158S-N218S, A088T-N109G-T158S-N218S-K256R-Q271H,A088T-N109G-T158S-N218S-N243V,A088T-N109G-T158S-N218S-N243V-K256R-Q275R,A088T-N109G-T158S-N218S-N243V-S248N-K256R-L257G,A088T-N109G-T158S-N218S-S248N, A088T-N109G-T158S-N218S-S248N-K256R,A088T-N109G-T158S-N218S-S248N-N269D, A088T-N109G-T158S-N243V-K256R,A088T-N109G-T158S-N243V-S248N-K256R,A088T-N109G-T158S-N243V-S248N-K256R-L257G-N269D,A088T-N109G-T158S-N243V-S248N-L257G,A088T-N109G-T158S-S248N-K256R-L257G, A088T-N109G-T158S-S248N-L257G,A088T-N109G-V147A-N218S-N243V-K256R, A088T-N218S, A088T-N218S-K256R,A088T-N218S-L257G-I268V, A088T-N218S-N243V, A088T-N218S-N243V-K256R,A088T-N218S-N243V-K256R-L257G, A088T-N218S-N243V-L257G,A088T-N218S-N243V-S248N-K256R-L257G, A088T-N218S-N243V-S248N-L257G,A088T-N218S-N243V-S248N-N269S, A088T-N218S-S248N-K256R,A088T-N243V-S248N, A088T-N243V-S248N-K256R, A088T-N243V-S248N-K256R,A088T-N243V-S248N-K256R-L257G, A088T-N243V-S248N-L257G, A088T-S248N,A088T-S248N, A088T-S248N-K256R-L257G, A088T-S248N-L257G,A088T-S248N-L257G-I268V, A088T-T158S, A088T-T158S, A088T-T158S-N218S,A088T-T158S-N218S-K256R, A088T-T158S-N218S-L257G,A088T-T158S-N218S-N243V-K256R-I268V,A088T-T158S-N218S-N243V-K256R-L257G,A088T-T158S-N218S-N243V-S248N-L257G, A088T-T158S-N218S-S248N,A088T-T158S-N243V-K256R, A088T-T158S-N243V-K256R-L257G,A088T-T158S-N243V-S248N-K256R, A088T-T158S-N243V-S248N-L257G,A088T-T158S-N243V-S248N-L257G, A088T-T158S-S248N,A088T-T158S-S248N-L257G, A088T-V147A-K256R,A098S-G131H-T158S-N218S-N243V-S248N-K256R-L257G,A116T-G131H-N218S-K256R, A116T-G131H-N218S-K256R-L257G,A116T-G131H-N218S-L257G, A116T-G131H-N218S-N243V-S248N-L257G,A116T-G131H-N218S-S248N-K256R-L257G, A116T-G131H-N243V-S248N,A116T-G131H-N243V-S248N-L257G, A116T-G131H-T158S-A231V-N243V-L257G,A116T-G131H-T158S-K256R, A116T-G131H-T158S-K256R-L257G,A116T-G131H-T158S-N218S-K256R, A116T-G131H-T158S-N218S-K256R-L257G,A116T-G131H-T158S-N218S-N243V, A116T-G131H-T158S-N218S-N243V-K256R,A116T-G131H-T158S-N218S-N243V-K256R-L257G,A116T-G131H-T158S-N218S-N243V-L257G,A116T-G131H-T158S-N218S-N243V-S248N-K256R,A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,A116T-G131H-T158S-N218S-N243V-S248N-L257G,A116T-G131H-T158S-N218S-S248N-K256R, A116T-G131H-T158S-N218T-L257G,A116T-G131H-T158S-S248N-K256R, A116T-G131H-T158S-S248N-L257G,A116T-N218S-K256R, A116T-N218S-K256R-L257G,A116T-N218S-N243V-S248N-K256R, A116T-N218S-N243V-S248N-K256R-L257G,A116T-N218S-N243V-S248N-L257G, A116T-N218S-S248N,A116T-N218S-S248N-K256R, A116T-N218S-S248N-L257G,A116T-N243V-S248N-L257G, A116T-S248N-L257G, A116T-T158S-L257G-Q271R,A116T-T158S-N218S-L257G, A116T-T158S-N218S-N243V-K256R-L257G,A116T-T158S-N218S-S248N-K256R, A116T-T158S-N218S-S248N-K256R-L257G,A116T-T158S-N243V-S248N-K256R, A116T-T158S-N243V-S248N-K256R-L257G,G024S-G053S-N078S-G097A-N101S-A128S, G131H, G131H-N218S,G131H-N218S-K256R, G131H-N218S-N243V-K256R,G131H-N218S-N243V-K256R-L257G, G131H-N218S-N243V-S248N,G131H-N218S-N243V-S248N-K256R, G131H-N218S-S248N-K256R-L257G,G131H-N218S-S248N-L257G, G131H-N243V-K256R, G131H-N243V-K256R-L257G,G131H-N243V-S248N-K256R-L257G, G131H-S248N-K256R, G131H-T158S-K256R,G131H-T158S-K256R-L257G, G131H-T158S-N218S-K256R,G131H-T158S-N218S-K256R-L257G, G131H-T158S-N218S-N243V-K256R-L257G,G131H-T158S-N218S-N243V-S248N, G131H-T158S-N218S-S248N-K256R-L257G,G131H-T158S-N218S-S248N-L257G, G131H-T158S-N243V-K256R,G131H-T158S-N243V-S248N-K256R, G131H-T158S-S248N-L257G,G131H-V147I-N218S-S248N-K256R,I107T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,I107T-N109G-G131H-N218S-N243V-K256R-L257G, N109G, N109G-A116T-G131H,N109G-A116T-G131H-A144V-T158S-S248N-K256R-L257G,N109G-A116T-G131H-K256R-L257G, N109G-A116T-G131H-L257G,N109G-A116T-G131H-N218S-N243V-K256R,N109G-A116T-G131H-N218S-N243V-S248N-K256R, N109G-A116T-G131H-N243V,N109G-A116T-G131H-N243V-K256R-L257G, N109G-A116T-G131H-N243V-S248N,N109G-A116T-G131H-N243V-S248N-K256R, N109G-A116T-G131H-S248N-K256R,N109G-A116T-G131H-T158S-K256R, N109G-A116T-G131H-T158S-K256R-L257G,N109G-A116T-G131H-T158S-L257G, N109G-A116T-G131H-T158S-N218S,N109G-A116T-G131H-T158S-N218S-K256R-L257G,N109G-A116T-G131H-T158S-N218S-L257G,N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,N109G-A116T-G131H-T158S-N218S-S248N-K256R,N109G-A116T-G131H-T158S-N218S-S248N-K256R-L257G,N109G-A116T-G131H-T158S-N243V-L257G,N109G-A116T-G131H-T158S-S248N-L257G,N109G-A116T-G131H-V147A-T158S-N218S-K256R-L257G,N109G-A116T-G131H-V149A-T158S-N218S-N243V-S248N-L257G,N109G-A116T-K256R, N109G-A116T-N218S-K256R, N109G-A116T-N218S-N243V,N109G-A116T-N218S-N243V-L257G, N109G-A116T-N218S-N243V-S248N-I268V,N109G-A116T-N218S-N243V-S248N-K256R-L257G,N109G-A116T-N218S-N243V-S248N-L257G, N109G-A116T-N243V-S248N,N109G-A116T-N243V-S248N-K256R, N109G-A116T-N243V-S248N-L257G,N109G-A116T-S248N-K256R, N109G-A116T-T158S,N109G-A116T-T158S-N218S-N243V-S248N-K256R,N109G-A116T-T158S-N218S-N243V-S248N-K256R-L257G,N109G-A116T-T158S-N218S-S248N-K256R, N109G-A116T-T158S-N243V-L257G,N109G-A116T-T158S-N243V-S248N, N109G-A116T-T158S-S248N,N109G-A116T-T158S-S248N-K256R, N109G-G131H, N109G-G131H-K256R,N109G-G131H-N218S-K256R, N109G-G131H-N218S-K256R-L257G,N109G-G131H-N218S-N243V-L257G, N109G-G131H-N218S-N243V-S248N-K256R,N109G-G131H-N218S-N243V-S248N-K256R-L257G,N109G-G131H-N218S-N243V-S248N-L257G, N109G-G131H-N218S-S248N-L257G,N109G-G131H-N243V, N109G-G131H-N243V-K256R, N109G-G131H-N243V-S248N,N109G-G131H-N243V-S248N-K256R-L257G, N109G-G131H-N243V-S248N-L257G,N109G-G131H-T158S-N218S-K256R-L257G, N109G-G131H-T158S-N218S-L257G,N109G-G131H-T158S-N218S-N243V,N109G-G131H-T158S-N218S-N243V-K256R-L257G,N109G-G131H-T158S-N218S-N243V-S248N,N109G-G131H-T158S-N218S-N243V-S248N-K256R-L257G,N109G-G131H-T158S-N218S-N243V-S248N-L257G,N109G-G131H-T158S-N218S-S248N-K256R-L257G,N109G-G131H-T158S-N243V-K256R-I268V, N109G-G131H-T158S-N243V-S248N,N109G-G131H-T158S-N243V-S248N-K256R,N109G-G131H-T158S-N243V-S248N-L257G, N109G-G131H-T158S-S248N,N109G-G131H-T158S-S248N-K256R-L257G, N109G-K141E-N218S-S248N-L257G,N109G-N218S, N109G-N218S-N243V-K256R, N109G-N218S-N243V-L257G,N109G-N218S-N243V-S248N-S260F, N109G-N218S-S248N,N109G-N218S-S248N-K256R, N109G-N243V-K256R, N109G-N243V-S248N,N109G-N243V-S248N-K256R, N109G-N243V-S248N-L257G,N109G-N243V-S248N-L257G-Q275R,N109G-S182F-S204F-S207L-N218S-S236F-S248N-L257G, N109G-S248N-K256R,N109G-T158S-K256R-L257G, N109G-T158S-L257G,N109G-T158S-N218S-N243V-K256R, N109G-T158S-N218S-N243V-S248N,N109G-T158S-N218S-N243V-S248N-L257G, N109G-T158S-N243V-K256R,N109G-T158S-N243V-S248N-K256R, N109G-T158S-N243V-S248N-L257G,N109G-T158S-S248N-L257G, N218S-N243V-L257G, N218S-N243V-S248N-K256R,N243V-K256R-L257G, N243V-S248N-L257G-Q271R,P057Q-A088T-N109G-A116T-G131H-T158S-N218S-S248N,S003P-A116T-N218S-K256R,S003P-N109G-G131H-N218S-N243V-S248N-K256R-L257G, S248N-K256R-L257G,T158S-K256R-L257G, T158S-N218S-A272V, T158S-N218S-K256R-L257G,T158S-N218S-L233S, T158S-N218S-N243V, T158S-N218S-N243V-K256R-L257G,T158S-N218S-N243V-L257G, T158S-N218S-N243V-S248N-K256R,T158S-N218S-S248N-K256R, T158S-N243V, T158S-N243V-K256R-L257G,T158S-N243V-S248N, T158S-N243V-S248N-K256R-N269D, andV004A-N109G-A116T-T158S-N218S-S248N-L257G, wherein amino acid positionsof the variant are numbered by correspondence with the sequence of SEQID NO:2. Such variants have enhanced proteolytic activity compared toBPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. Theinvention includes a protease variant having enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2), a PI value of 1.0 relative toBPN′-v3, and a PI value of 1.0 relative to BPN′-v36 in this assay, thevariant comprising an amino acid sequence having at least 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity toSEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto about 0.9 relative to BPN′-v36 in a BMI microswatch cleaning assay(Test Method 3) in Detergent Composition 4 at pH 8 and 16° C.:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of A088T-A098S-N218S-K256R,A088T-A116T-G131H-K256R, A088T-A116T-G131H-K256R-L257G-L267M,A088T-A116T-G131H-N218S-N243V-K256R,A088T-A116T-G131H-N218S-N243V-K256R-L257G,A088T-A116T-G131H-N218S-N243V-S248N, A088T-A116T-G131H-N218S-S248N,A088T-A116T-G131H-N218S-S248N-K256R,A088T-A116T-G131H-N218S-S248N-K256R, A088T-A116T-G131H-N243V,A088T-A116T-G131H-S248N, A088T-A116T-G131H-S248N-L257G,A088T-A116T-G131H-S248N-L257G, A088T-A116T-G131H-T158S-N218S,A088T-A116T-G131H-T158S-N218S-K256R-L257G,A088T-A116T-G131H-T158S-N218S-N243V-S248N-K256R,A088T-A116T-G131H-T158S-N218S-N243V-S248N-L257G,A088T-A116T-G131H-T158S-N218S-S248N,A088T-A116T-G131H-T158S-N218S-S248N-K256R, A088T-A116T-K256R-L257G,A088T-A116T-N218S-I268V, A088T-A116T-N218S-K256R,A088T-A116T-N218S-N243V-Q271R, A088T-A116T-N218S-N243V-S248N-K256R,A088T-A116T-N218S-N243V-S248N-K256R-Q275R, A088T-A116T-N218S-S248N,A088T-A116T-N218S-S248N-K256R, A088T-A116T-N243V-S248N-K256R,A088T-A116T-T158S, A088T-A116T-T158S-N218S-K256R,A088T-A116T-T158S-N218S-N243V-L257G,A088T-A116T-T158S-N218S-N243V-S248N-L257G,A088T-A116T-T158S-N218S-S248N, A088T-A116T-T158S-N218S-S248N-K256R,A088T-A116T-T158S-N218S-S248N-K256R-L257G,A088T-A116T-T158S-N218S-S248N-L257G, A088T-A116T-T158S-S248N-L257G,A088T-G131H, A088T-G131H, A088T-G131H-N218S-K237R-K256R-L257G,A088T-G131H-N218S-K256R, A088T-G131H-N218S-K256R-L257G,A088T-G131H-N218S-N243V-K256R-L257G, A088T-G131H-N218S-N243V-L257G,A088T-G131H-N218S-N243V-L257G, A088T-G131H-N218S-N243V-S248N,A088T-G131H-N218S-N243V-S248N-K256R,A088T-G131H-N218S-N243V-S248N-K256R-L257G,A088T-G131H-N218S-N243V-S248N-K256R-L257G, A088T-G131H-N218S-S248N,A088T-G131H-N243V, A088T-G131H-N243V-K256R,A088T-G131H-N243V-K256R-L257G, A088T-G131H-N243V-S248N,A088T-G131H-N243V-S248N, A088T-G131H-N243V-S248N-K256R,A088T-G131H-S248N, A088T-G131H-S248N-K256R,A088T-G131H-T158S-N218S-K256R-L257G, A088T-G131H-T158S-N218S-L257G,A088T-G131H-T158S-N218S-N243V,A088T-G131H-T158S-N218S-N243V-K256R-L257G,A088T-G131H-T158S-N218S-N243V-S248N-K256R,A088T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,A088T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,A088T-G131H-T158S-N218S-N243V-S248N-L257G,A088T-G131H-T158S-N218S-S248N-K256R,A088T-G131H-T158S-N218S-S248N-K256R-L257G,A088T-G131H-T158S-S248N-K256R-L257G, A088T-L257G,A088T-N109G-A116T-G131H-N218S-L257G,A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R,A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R-L257G,A088T-N109G-A116T-G131H-N218S-N243V-S248N-N269D,A088T-N109G-A116T-G131H-N218S-N243V-S248N-Q275R,A088T-N109G-A116T-G131H-N218S-S248N-K256R-L257G,A088T-N109G-A116T-G131H-N243V-S248N, A088T-N109G-A116T-G131H-T158S,A088T-N109G-A116T-G131H-T158S-N218S-L257G-I268V,A088T-N109G-A116T-G131H-T158S-N218S-N243V-K256R,A088T-N109G-A116T-G131H-T158S-N218S-N243V-K256R-L257G,A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N,A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-L257G,A088T-N109G-A116T-G131H-T158S-N218S-S248N-L257G,A088T-N109G-A116T-G131H-T158S-S248N,A088T-N109G-A116T-G131H-T158S-S248N,A088T-N109G-A116T-G131H-W241L-S248N-K256R-L257G,A088T-N109G-A116T-K256R, A088T-N109G-A116T-N218S-K256R-L257G,A088T-N109G-A116T-N218S-N243V-S248N-K256R,A088T-N109G-A116T-N218S-S248N, A088T-N109G-A116T-T158S-N218S,A088T-N109G-A116T-T158S-N218S-K256R-L257G,A088T-N109G-A116T-T158S-N218S-N243V-S248N-L257G,A088T-N109G-A116T-T158S-N243V-S248N,A088T-N109G-A116T-T158S-N243V-S248N-K256R,A088T-N109G-G131H-A138V-T158S-N218S-N243V-S248N-L257G,A088T-N109G-G131H-K256R-L257G, A088T-N109G-G131H-N218S-N243V,A088T-N109G-G131H-N218S-N243V-S248N-L257G,A088T-N109G-G131H-N218S-S248N-K256R-L257G,A088T-N109G-G131H-N218S-S248N-K256R-L257G-Q275R,A088T-N109G-G131H-N243V-S248N,A088T-N109G-G131H-N243V-S248N-K256R-L257G, A088T-N109G-G131H-T158S,A088T-N109G-G131H-T158S-L233S-N243V-S248N,A088T-N109G-G131H-T158S-N218S-N243V-K256R,A088T-N109G-G131H-T158S-N218S-N243V-K256R-L257G,A088T-N109G-G131H-T158S-N218S-N243V-S248N-K256R-L257G,A088T-N109G-G131H-T158S-N218S-N243V-S248N-K256R-L257G,A088T-N109G-G131H-T158S-N218S-N243V-S248N-L257G,A088T-N109G-G131H-T158S-N218S-S248N-K256R,A088T-N109G-G131H-T158S-N243V-S248N-K256R,A088T-N109G-G131H-T158S-S248N-K256R,A088T-N109G-G131H-T158S-S248N-K256R-L257G,A088T-N109G-G131H-V149A-K256R-L257G, A088T-N109G-N218S-K256R-L257G,A088T-N109G-N218S-N243V-K256R, A088T-N109G-N218S-N243V-L257G,A088T-N109G-N218S-N243V-S248N,A088T-N109G-N218S-N243V-S248N-K256R-L257G,A088T-N109G-N218S-S248N-K256R, A088T-N109G-N243V-K256R,A088T-N109G-N243V-S248N-K256R, A088T-N109G-S248N-K256R-L257G,A088T-N109G-T158S, A088T-N109G-T158S-K256R-L257G,A088T-N109G-T158S-N218S-N243V-K256R,A088T-N109G-T158S-N218S-N243V-K256R-L257G,A088T-N109G-T158S-N218S-N243V-S248N-K256R,A088T-N109G-T158S-N218S-N243V-S248N-L257G,A088T-N109G-T158S-N218S-S248N, A088T-N109G-T158S-S248N,A088T-N109G-T158S-S248N, A088T-N218S-N243V-K256R,A088T-N218S-N243V-L257G, A088T-N218S-N243V-S248N,A088T-N218S-N243V-S248N-K256R, A088T-N218S-N243V-S248N-K256R,A088T-N218S-S248N, A088T-N218S-S248N-L257G, A088T-S248N-K256R-L257G,A088T-T158S-K256R, A088T-T158S-N218S-N243V-K256R,A088T-T158S-N218S-N243V-L257G, A088T-T158S-N218S-N243V-S248N,A088T-T158S-N218S-N243V-S248N-K256R,A088T-T158S-N218S-N243V-S248N-K256R-L257G,A088T-T158S-N218S-N243V-S248N-K256R-L257G,A088T-T158S-N218S-N243V-S248N-L257G, A088T-T158S-N218S-S248N,A088T-T158S-N218S-S248N-K256R, A088T-T158S-N218S-S248N-L257G,A088T-T158S-N218S-S248N-L257G-Q275K, A088T-T158S-N243V,A088T-T158S-N243V-K256R, A088T-T158S-N243V-S248N,A088T-T158S-S248N-K256R-L257G, A088T-T158S-S248N-L257G,A088T-V147I-N218S-N243V-K256R-L257G, A116T-G131H-L257G,A116T-G131H-N218S-N243V, A116T-G131H-N218S-N243V-K256R-L257G,A116T-G131H-N218S-N243V-S248N-K256R-L257G, A116T-G131H-N218S-S248N,A116T-G131H-N218S-S248N-K256R, A116T-G131H-N243V-S248N-K256R,A116T-G131H-T158S-N218S-N243V-S248N, A116T-G131H-T158S-N218S-S248N,A116T-G131H-T158S-N243V-K256R, A116T-G131H-V130I-N218S-N243V-S248N,A116T-K141E-N218S-N243V-S248N-K256R-L257G, A116T-K256R,A116T-N218S-N243V-S248N, A116T-N218T-N243V-S248N,A116T-N243V-K256R-L257G, A116T-S248N-K256R, A116T-T158S-N218S-K256R,A116T-T158S-N218S-K256R-L257G, A116T-T158S-N218S-N243V-L257G,A116T-T158S-N218S-N243V-S248N, A116T-T158S-N218S-S248N-L257G,G024S-G053S-N078S-G097A-N101S,G053S-A088T-N109G-A116T-G131H-T158S-G169S-N218S-S248N-K256R-L257G,G131H-K141R-T158S-N218S-K256R, G131H-K256R, G131H-N218S-K256R-L257G,G131H-N218S-N243V-S248N-L257G, G131H-N218S-S248N-K256R,G131H-N243V-S248N, G131H-N243V-S248N-L257G, G131H-T158S-N218S,G131H-T158S-N218S-N240H-N243V-S248N-K256R-L257G,G131H-T158S-N218S-N243V, G131H-T158S-N218S-S248N-K256R-L257G-N269S,G131H-T158S-N243V-L257G, G131H-T158S-N243V-S248N, G131H-T158S-S248N,K256R, K256R-L257G, N109G-A116T-G131H-N218S-N243V,N109G-A116T-G131H-N218S-N243V-L257G,N109G-A116T-G131H-N218S-S248N-L257G,N109G-A116T-G131H-N218S-W241R-N243V-K256R,N109G-A116T-G131H-S248N-L257G, N109G-A116T-G131H-T158S-N218S-K256R,N109G-A116T-G131H-T158S-N218S-N243V-K256R-L257G,N109G-A116T-G131H-T158S-N218S-N243V-S248N,N109G-A116T-G131H-T158S-N218S-N243V-S248N-L257G,N109G-A116T-G131H-T158S-N243V-K256R-L257G,N109G-A116T-G131H-T158S-N243V-S248N-K256R,N109G-A116T-G131H-T158S-S248N-K256R-L257G,N109G-A116T-I234T-N243V-S248N-K256R-L257G,N109G-A116T-N218S-N243V-S248N, N109G-A116T-N243V-K256R-L257G,N109G-A116T-T158S-N218S-K237R-N243V-S248N,N109G-G131H-N218S-N243V-S248N, N109G-G131H-S248N, N109G-G131H-T158S,N109G-G131H-T158S-L257G, N109G-G131H-T158S-N218S-N243V-S248N-K256R,N109G-G131H-T158S-N218S-S248N-K256R,N109G-G131H-T158S-N218S-S248N-L257G,N109G-G131H-T158S-N243V-S248N-K256R-L257G,N109G-G131H-T158S-S248N-K256R, N109G-N218S-K256R-L257G,N109G-N218S-N243V-S248N-K256R, N109G-N218S-S248N-L257G, N109G-S248N,N109G-T158S-N218S, N109G-T158S-N218S-N243V, N109G-T158S-N243V-L257G,N218S-K256R, N218S-N243V-K256R, N218S-N243V-S248N, N218S-S248N,N218S-S248N-K256R, N243V-L257G,S003P-N109G-A116T-G131H-N218S-N243V-S248N,S003P-N109G-A116T-G131H-T158S-N218S-K256R,S105H-W106G-I107L-I108S-N109A-G110A-I111S-E112N-W113G-A114P,S248N-L257G, T158S, T158S-K256R, T158S-N218S, T158S-N218S-K256R,T158S-N218S-L233S-S248N, T158S-N218S-L257G, T158S-N218S-N243V-K256R,T158S-N218S-N243V-S248N, T158S-N218S-N243V-S248N-L257G,T158S-N218S-S248N-K256R-L257G, T158S-N218S-S248N-L257G,T158S-N243V-S248N-L257G, T158S-S248N, andV004L-A088T-G131H-T158S-N218S-S248N-L257G, wherein amino acid positionsof the variant are numbered by correspondence with the sequence of SEQID NO:2. Such variants have proteolytic activity. The invention includesa protease variant having proteolytic activity and/or a PI value of 0.9relative to BPN′-v36 in this assay, the variant comprising an amino acidsequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 andcomprising at least one set of acid substitutions selected from saidgroup above, wherein amino acid positions of the variant are numbered bycorrespondence with amino acid positions of the SEQ ID NO:2 sequence.Also included are compositions, including, but not limited to, e.g.,cleaning compositions, comprising at least one such variant and methodsfor cleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto or greater than 0.5 and less than 0.9 relative to BPN′-v36 in a BMImicroswatch cleaning assay (Test Method 3) in Detergent Composition 4 atpH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acidsequence (SEQ ID NO:6) comprising at least one set of amino acidsubstitutions selected from the group consisting ofA015S-A088T-N109G-G131H-T158S-N218S-S248N,A088T-A098S-G131H-S248N-K256R-L257G,A088T-A116T-G131H-N218S-N243V-K256R-L257G,A088T-A116T-G131H-T158S-L257G, A088T-A116T-G131H-T158S-N218S-L257G,A088T-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,A088T-A116T-G131H-T158S-N218S-N243V-S248N-L257G,A088T-A116T-N218S-L257G, A088T-A116T-T158S-K256R,A088T-A116T-T158S-S248N-K256R-L257G,A088T-G131H-K141E-N218S-N243V-S248N-L257G, A088T-G131H-K256R,A088T-G131H-N218S-K256R, A088T-G131H-N218S-N243V-S248N-K256R,A088T-G131H-N218S-N243V-S248N-L257G, A088T-G131H-N218S-S248N-K256R,A088T-G131H-N218S-S248N-K256R, A088T-G131H-T158S-S248N-K256R,A088T-G131H-T158S-S248N-K256R-L257G,A088T-I107T-N109G-G131H-N218S-S248N-K256R,A088T-N109G-A116T-G131H-D140G-T158S-N218S-N243V-K256R,A088T-N109G-A116T-G131H-N218S-N243V-K256R,A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R,A088T-N109G-A116T-G131H-T158S-N218S-S248N-L257G,A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R-I268V,A088T-N109G-A116T-G131H-V149A-N218S-S248N-K256R-L257G,A088T-N109G-A116T-N218S-N243V-S248N-K256R-L257G,A088T-N109G-A116T-T158S-K256R-L257G,A088T-N109G-A116T-T158S-N218S-N243V-L257G, A088T-N109G-D140G-N243V,A088T-N109G-G131H-D140G-T158S-N243V-S248N-K256R,A088T-N109G-G131H-K141E-T158S-N218S-K256R,A088T-N109G-G131H-N218S-S248N,A088T-N109G-G131H-N218S-S248N-K256R-Q271R,A088T-N109G-G131H-N218S-S248N-L257G, A088T-N109G-G131H-T158S-K256R,A088T-N109G-G131H-T158S-N218S-S248N-K256R,A088T-N109G-G131H-V149L-T158S-K256R-L257G, A088T-N109G-T158S-N218S,A088T-N109G-T158S-N218S-K256R-L257G-Q271K,A088T-N109G-T158S-N218S-L257G, A088T-N109G-T158S-S248N-K256R,A088T-N218S-S248N-L257G-Q271R, A088T-T158S-N218S-K256R-L257G,A088T-T158S-N218S-N243V-K256R, A088T-Y104H-A116T-G131H-N218S-N243V,A116T-G131H-K141E-N218S-N243V-S248N-L257G,A116T-G131H-N218S-N243V-S248N-K256R,A116T-G131H-T158S-N218S-S248N-L257G-N269D,A116T-G131H-T158S-N218S-S248N-Q271R, A116T-G131H-T158S-N243V-S248N,A116T-G157E-T158S-N243V-S248N-K256R, A116T-T158S-N218S,G131H-N218S-L257G, G131H-N218S-S248N,G131H-T158S-N218S-N243V-S248N-K256R-L257G,G131H-T158S-N218S-N243V-S248N-L257G, G131H-T158S-N218S-S248N-I268V,I107T-N109G-G131H-N218S-L257G, L090I-N109G-T158S-N243V, L257G,N109G-A116T-G131H-T158S-N218S-K256R-L257G-Q271R,N109G-A116T-N218S-W241R-N243V-S248N-K256R-L257G,N109G-G131H-K141E-L257G, N109G-G131H-N218S-N243V,N109G-T158S-N218S-N243V-L257G, N109G-T158S-N218S-S248N-K256R,N109G-T158S-N243V-S248N-K256R-L257G, N218S-S248N-K256R-L257G,S003P-N109G-G131H-T158S-L257G, S003P-S248N-L257G,T158S-S248N-K256R-L257G, V004A-A088T-G131H-N218S-N243V-S248N-L257G,Y006H-N218S-N243V-S248N, Y104H-N109G-G131H-N243V-S248N,A088T-A116T-T158S-N218S-N243V-S248N-K256R, A088T-A116T-T158S-N243V,A088T-G131H-T158S-N218S-I234T-S248N-L257G,A088T-G131H-T158S-N218S-N243V-S248N-K256R,A088T-G131H-V149L-T158S-N243V-S248N-K256R-L257G,A088T-I107T-N109G-G131H-N218S-A223G-S248N-K256R,A088T-K213N-N243V-S248N-K256R, A088T-K256R-L257G,A088T-N109G-A116T-G131H-A232S-N243V-K256R,A088T-N109G-A116T-G131H-D140G-S248N-L257G,A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R-L257G,A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N,A088T-N109G-A116T-G131H-T158S-N243V-S248N-L257G,A088T-N109G-A116T-M124I-G131H-T158S-N218S-S248N-L257G,A088T-N109G-A116T-V148A-N218S-N243V,A088T-N109G-G131H-N218S-N243V-S248N,A088T-N109G-N218S-S248N-T255K-K256R-L257G, A088T-T158S-N218S-L257G,A088T-T158S-N218S-Q245K-S248N-K256R, A088T-T158S-N218S-S248N-K256R,A116T-G131H-N218S-N243V-K256R,A116T-G131H-N218S-W241R-N243V-S248N-K256R-L257G,A116T-G131H-T158S-N218S-L257G,A116T-G131H-V150A-T158S-N243V-S248N-K256R-L257G,I107T-G131H-T158S-N243V-S248N-K256R-L257G,N109G-A116T-K141E-T158S-N218S-N243V-L257G,N109G-A116T-T158S-N218S-N243V-S248N, T158S-N243V-S248N-K256R,T158S-N243V-S248N-K256R-L257G, A088T-A116T-G131H-G146C,A088T-A116T-N218S, A088T-A116T-T158S-N243V-K256R-L257G,A088T-A138E-N218S-N243V-K256R,A088T-N109G-A116T-G131H-T158S-N218S-N243F-S248N,A088T-T158S-V203I-N218S-K256R-L257G,A116T-D140G-T158S-N218S-N243V-S248N, A088T-A116T-T158S-K256R-L257G,A088T-A116T-T158S-N218S-N243V-S248N-E251K-K256R-L257G,A088T-I108T-N109G-G131H-T158S-N218S-S248N-K256R-L257G,A088T-N109G-A116T-G131H-K141E-N218S, A088T-N109G-W241R-S248N-K256R, andG065D-A088T-G131H-N243V-S248N, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have proteolytic activity. The invention includes aprotease variant having proteolytic activity and/or a PI value equal toor greater than 0.5 and less than 0.9 relative to BPN′-v36 in thisassay, the variant comprising an amino acid sequence having at least60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least oneset of acid substitutions selected from said group above, wherein aminoacid positions of the variant are numbered by correspondence with aminoacid positions of the SEQ ID NO:2 sequence. Also included arecompositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI valuegreater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from greater than 1.0 to about 10, from greater than1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence(SEQ ID NO:6) comprising at least one set of amino acid substitutionsselected from the group consisting ofA088T-N109G-A116T-T158S-N243V-L257G, A116T-N218S-N243V-L257G-N269S,A088T-A116T-K256R, A088T-G131H-K256R,A088T-N109G-A116T-T158S-S248N-K256R-L257G, A088T-N109G-T158S-L257G,A088T-A116T-G131H-T158S-N218S-N243V-K256R-A273T,A088T-A116T-N243V-L257G, A088T-A116T-S248N-K256R-L257G,A088T-A116T-T158S-N243V-L257G, A088T-A116T-T158S-N243V-S248N-L257G,A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R-L257G,A088T-N109G-A116T-G131H-N243V-L257G,A088T-N109G-A116T-G131H-T158S-L257G,A088T-N109G-A116T-T158S-N218S-L257G,A088T-N109G-A116T-T158S-N218S-N243V-S248N-K256R-L257G,A088T-N109G-G131H-N218S-K256R-L257G, A088T-N109G-N218S-S248N-L257G,A088T-T158S-N218S-N243V-K256R-I268V, A088T-T158S-N218S-S248N-L257G,A116T-N218S-K256R-L257G, N109G-A116T, N109G-A116T-G131H-T158S-L257G,N109G-A116T-N243V, N109G-A116T-N243V-K256R, N109G-A116T-T158S-L257G,N109G-K256R, N109G-N243V-K256R-L257G, S003P-N109G-G131H-T158S-K256R,A088T-A116T, A088T-A116T-G131H-N218S-K256R-L257G,A088T-A116T-G131H-N218S-L257G,A088T-A116T-G131H-N218S-N243V-S248N-L257G,A088T-A116T-G131H-N243V-K256R-L257G,A088T-A116T-G131H-N243V-S248N-K256R-L257G,A088T-A116T-G131H-T158S-N218S-N243V,A088T-A116T-G131H-T158S-N218S-N243V-K256R-L257G,A088T-A116T-G131H-T158S-N218S-N243V-S248N,A088T-A116T-G131H-T158S-S248N-K256R-L257G,A088T-A116T-G131H-T158S-S248N-L257G, A088T-A116T-N218S-N243V-L257G,A088T-A116T-N218S-N243V-S248N-K256R-L257G,A088T-A116T-N218S-N243V-S248N-K256R-Q275R,A088T-A116T-T158S-A216S-N218S-N243V-K256R-L257G,A088T-A116T-T158S-K256R, A088T-A116T-T158S-N218S-L257G,A088T-A116T-T158S-N218S-N243V, A088T-A116T-T158S-N218S-N243V-K256R,A088T-A116T-T158S-N218S-N243V-K256R-L257G,A088T-A116T-T158S-N218S-N243V-K256R-N269S, A088T-A116T-T158S-N243V,A088T-A116T-T158S-N243V-K256R,A088T-A116T-V147I-T158S-N218S-N243V-L257G, A088T-G131H-K256R-L257G,A088T-G131H-N218S-N243V-S248N-K256R-L257G,A088T-G131H-S248N-K256R-L257G, A088T-G131H-T158S-N218S-L257G,A088T-G131H-T158S-N218S-N243V-L257G,A088T-I107T-N109G-A116T-G131H-T158S-N218S-N243V-S248N,A088T-I107T-N109G-G131H-N218S-S248N-K256R,A088T-N109G-A116T-G131H-A153S-N218S-S248N-L257G,A088T-N109G-A116T-G131H-K256R-L257G,A088T-N109G-A116T-G131H-N218S-K256R-L257G,A088T-N109G-A116T-G131H-N218S-L257G,A088T-N109G-A116T-G131H-N218S-N243V-K256R,A088T-N109G-A116T-G131H-N218S-N243V-L257G,A088T-N109G-A116T-G131H-N243V-L257G,A088T-N109G-A116T-G131H-S248N-L257G,A088T-N109G-A116T-G131H-T158S-N218S,A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R,A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-L257G,A088T-N109G-A116T-N218S-K256R-L257G, A088T-N109G-A116T-N218S-L257G,A088T-N109G-A116T-N218S-N243V, A088T-N109G-A116T-N218S-N243V-L257G,A088T-N109G-A116T-N218T-K256R, A088T-N109G-A116T-N218T-K256R-L257G,A088T-N109G-A116T-N243V, A088T-N109G-A116T-N243V-K256R-L257G,A088T-N109G-A116T-N243V-K256R-L257G-N269D, A088T-N109G-A116T-T158S,A088T-N109G-A116T-T158S, 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A088T-N109G-A116T-T158S-S248N,A088T-N109G-A116T-T158S-S248N-K256R,A088T-N109G-A116T-T158S-S248N-L257G, A088T-N109G-G131H-N218S-K256R,A088T-N109G-G131H-N218S-K256R, A088T-N109G-G131H-N218S-N243V-K256R,A088T-N109G-G131H-N218S-N243V-K256R,A088T-N109G-G131H-N218S-N243V-K256R-L257G,A088T-N109G-G131H-N218S-N243V-S248N-K256R,A088T-N109G-G131H-N218S-S248N, A088T-N109G-G131H-N218S-S248N-L257G,A088T-N109G-G131H-N243V-K256R-L257G,A088T-N109G-G131H-N243V-K256R-L257G, A088T-N109G-G131H-N243V-L257G,A088T-N109G-G131H-N243V-L257G, A088T-N109G-G131H-N243V-S248N-K256R,A088T-N109G-G131H-N243V-S248N-K256R-L257G,A088T-N109G-G131H-N243V-S248N-L257G, A088T-N109G-G131H-S248N-L257G,A088T-N109G-G131H-T158S-K256R-L257G,A088T-N109G-G131H-T158S-N218S-L257G,A088T-N109G-G131H-T158S-N218S-L257G,A088T-N109G-G131H-T158S-N218S-N243V-S248N,A088T-N109G-G131H-T158S-N218S-N243V-S248N-K256R,A088T-N109G-G131H-T158S-N218S-S248N-K256R,A088T-N109G-G131H-T158S-N218S-S248N-L257G,A088T-N109G-G131H-T158S-N243V, A088T-N109G-G131H-T158S-N243V-S248N,A088T-N109G-G131H-T158S-N243V-S248N-K256R,A088T-N109G-G131H-T158S-N243V-S248N-K256R,A088T-N109G-G131H-T158S-N243V-S248N-K256R-L257G,A088T-N109G-G131H-T158S-S248N-K256R-L257G,A088T-N109G-G131H-T158S-W241R-S248N-K256R,A088T-N109G-G131H-V148A-N218S-N243V-K256R-L257G,A088T-N109G-G154A-N155P-E156T-G157L-T158M-S159E-G160E-S161L,A088T-N109G-N218S-K256R, A088T-N109G-N218S-N243V-L257G,A088T-N109G-N218S-N243V-S248N, A088T-N109G-N243V-K256R,A088T-N109G-N243V-S248N, A088T-N109G-N243V-S248N-K256R-L257G,A088T-N109G-S248N, A088T-N109G-T158S-N218S,A088T-N109G-T158S-N218S-K256R, A088T-N109G-T158S-N218S-K256R-L257G,A088T-N109G-T158S-N218S-L257G,A088T-N109G-T158S-N218S-N243V-S248N-K256R,A088T-N109G-T158S-N218S-N243V-S248N-K256R-L257G,A088T-N109G-T158S-N218S-S248N-K256R,A088T-N109G-T158S-N218S-S248N-K256R-L257G,A088T-N109G-T158S-N243V-K256R, A088T-N109G-T158S-N243V-K256R,A088T-N109G-T158S-N243V-S248N-L257G,A088T-N109G-T158S-S248N-K256R-L257G, A088T-N109G-T158S-S248N-L257G,A088T-N109G-T158S-S248N-L257G, A088T-N109G-V147A-N218S-N243V-K256R,A088T-N218S-L257G-I268V, A088T-N218S-N243V, A088T-N218S-N243V-S248N,A088T-N218S-N243V-S248N-N269S, A088T-N218S-S248N-K256R,A088T-N218S-S248N-L257G-Q271R, A088T-N243V, A088T-N243V,A088T-N243V-K256R, A088T-N243V-S248N-K256R,A088T-N243V-S248N-K256R-L257G, A088T-S248N,A088T-T158S-N218S-K256R-L257G, A088T-T158S-N218S-N243V-K256R,A088T-T158S-N218S-N243V-L257G,A088T-T158S-N218S-N243V-S248N-K256R-L257G,A088T-T158S-N218S-N243V-S248N-K256R-L257G,A088T-T158S-N243V-K256R-L257G, A088T-T158S-N243V-K256R-L257G-Q271H,A088T-T158S-N243V-S248N, A088T-T158S-N243V-S248N-L257G,A088T-T158S-S248N, A088T-V147A-K256R, A116T-G131H-K256R,A116T-G131H-N218S, A116T-G131H-N218S-K256R-L257G,A116T-G131H-N218S-L257G, A116T-G131H-N218S-N243V,A116T-G131H-N218S-S248N-K256R, A116T-G131H-N218S-S248N-K256R-L257G,A116T-G131H-N243V-S248N, A116T-G131H-N243V-S248N-L257G,A116T-G131H-S248N-K256R, A116T-G131H-T158S-A231V-N243V-L257G,A116T-G131H-T158S-N218S-K256R, A116T-G131H-T158S-N218S-K256R-L257G,A116T-G131H-T158S-N218S-N243V-K256R-L257G,A116T-G131H-T158S-N218S-N243V-S248N-K256R,A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,A116T-G131H-T158S-N218S-S248N, A116T-G131H-T158S-N243V-L257G,A116T-G131H-T158S-N243V-S248N-K256R, A116T-G131H-T158S-S248N-K256R,A116T-G131H-T158S-S248N-L257G, A116T-G131H-V143F-T158S-N218S,A116T-K256R, A116T-N218S, A116T-N218S-K256R, A116T-N218S-N243V-L257G,A116T-N218S-N243V-S248N-K256R, A116T-N218S-N243V-S248N-K256R-L257G,A116T-N218S-N243V-S248N-L257G, A116T-N218S-S248N-L257G, A116T-N243V,A116T-N243V-K256R-L257G, A116T-N243V-S248N,A116T-N243V-S248N-K256R-L257G, A116T-S248N-K256R-L257G,A116T-T158S-K256R-L257G, A116T-T158S-N218S, A116T-T158S-N218S-K256R,A116T-T158S-N218S-N243V, A116T-T158S-N218S-N243V-S248N,A116T-T158S-N218S-S248N-K256R, A116T-T158S-N218S-S248N-K256R-L257G,A116T-T158S-N243V-K256R, A116T-T158S-N243V-L257G,A116T-T158S-N243V-S248N, A116T-T158S-S248N-K256R-L257G,G131H-K141R-T158S-N218S-K256R, G131H-N218S, G131H-N218S-K256R,G131H-N218S-N243V-K256R-L257G, G131H-N218S-N243V-S248N,G131H-N218S-N243V-S248N-L257G, G131H-N243V-S248N-K256R,G131H-N243V-S248N-K256R-L257G, G131H-S248N,G131H-T158S-I234T-N243V-K256R, G131H-T158S-N218S-K256R-L257G,G131H-T158S-N218S-N243V, G131H-T158S-N218S-N243V-S248N-L257G,G131H-T158S-N218S-S248N-K256R-L257G, G131H-T158S-N218S-S248N-L257G,G131H-T158S-N243V-K256R, G131H-T158S-N243V-S248N-L257G, N109G,N109G-A116T-G131H-A144V-T158S-S248N-K256R-L257G,N109G-A116T-G131H-K256R-L257G, N109G-A116T-G131H-N218S-N243V-K256R,N109G-A116T-G131H-N218S-N243V-K256R-L257G,N109G-A116T-G131H-N218S-S248N-K256R,N109G-A116T-G131H-N218S-S248N-L257G, N109G-A116T-G131H-N243V-K256R,N109G-A116T-G131H-N243V-S248N,N109G-A116T-G131H-N243V-S248N-K256R-L257G, N109G-A116T-G131H-S248N,N109G-A116T-G131H-S248N-K256R, N109G-A116T-G131H-T158S-K256R,N109G-A116T-G131H-T158S-K256R-L257G,N109G-A116T-G131H-T158S-N218S-K256R,N109G-A116T-G131H-T158S-N218S-K256R-L257G,N109G-A116T-G131H-T158S-N218S-L257G,N109G-A116T-G131H-T158S-N218S-N243V-K256R,N109G-A116T-G131H-T158S-N218S-N243V-S248N-L257G,N109G-A116T-G131H-T158S-N218S-S248N,N109G-A116T-G131H-T158S-N243V-L257G,N109G-A116T-G131H-T158S-N243V-S248N, N109G-A116T-G131H-T158S-S248N,N109G-A116T-G131H-T158S-S248N-K256R,N109G-A116T-G131H-T158S-S248N-K256R-L257G,N109G-A116T-G131H-V149A-T158S-N218S-N243V-S248N-L257G,N109G-A116T-N218S, N109G-A116T-N218S-K256R,N109G-A116T-N218S-N243V-K256R-L257G,N109G-A116T-N218S-N243V-S248N-I268V, N109G-A116T-N243V-K256R-L257G,N109G-A116T-N243V-S248N, N109G-A116T-N243V-S248N-L257G,N109G-A116T-T158S, N109G-A116T-T158S-N218S-N243V-K256R-L257G,N109G-A116T-T158S-N218S-N243V-S248N-K256R-L257G,N109G-A116T-T158S-N218S-N243V-S248N-L257G,N109G-A116T-T158S-N218S-S248N-K256R-L257G,N109G-A116T-T158S-N243V-K256R-L257G, N109G-A116T-T158S-N243V-L257G,N109G-A116T-T158S-N243V-S248N-L257G, N109G-A116T-T158S-Q275R,N109G-A116T-T158S-S248N-K256R-L257G, N109G-G131H-L257G,N109G-G131H-N218S-N243V-S248N-K256R-L257G,N109G-G131H-N218S-N243V-S248N-L257G,N109G-G131H-N218S-S248N-K256R-L257G, N109G-G131H-N243V-K256R,N109G-G131H-S145F-N218S-N243V-K256R-L257G,N109G-G131H-S248N-K256R-L257G, N109G-G131H-S248N-L257G,N109G-G131H-T158S-K256R, N109G-G131H-T158S-N218S-L257G,N109G-G131H-T158S-N218S-N243V, N109G-G131H-T158S-N218S-N243V-K256R,N109G-G131H-T158S-N218S-N243V-K256R-L257G,N109G-G131H-T158S-N218S-N243V-S248N-L257G,N109G-G131H-T158S-N218S-S248N-K256R,N109G-G131H-T158S-N218S-S248N-K256R-L257G-A274T,N109G-G131H-T158S-N218S-S248N-L257G, N109G-G131H-T158S-N243V-S248N,N109G-G131H-T158S-N243V-S248N-K256R-L257G,N109G-G131H-T158S-S248N-K256R-L257G, N109G-G131H-T158S-S248N-L257G,N109G-N218S-K256R-L257G, N109G-N218S-L257G, N109G-N218S-N243V-K256R,N109G-N218S-N243V-S248N-S260F, N109G-N218S-S248N, N109G-N243V-K256R,N109G-N243V-L257G, N109G-N243V-S248N, N109G-N243V-S248N-K256R-L257G,N109G-S182F-S204F-S207L-N218S-S236F-S248N-L257G, N109G-S248N-K256R,N109G-T158S-K256R, N109G-T158S-N218S-N243V-K256R-L257G,N109G-T158S-N218S-S248N-L257G, N109G-T158S-N243V,N109G-T158S-N243V-K256R, N109G-T158S-N243V-S248N,N109G-T158S-N243V-S248N-K256R, N109G-T158S-S248N-K256R,N109G-T158S-S248N-L257G, N218S, N218S-N243V-S248N-K256R,N218S-S248N-L257G, N243V-K256R, N243V-S248N-K256R,N243V-S248N-K256R-L257G, N243V-S248N-L257G-Q271R,S003P-A116T-T158S-S248N-K256R, S248N, T158S-N218S, T158S-N218S-A272V,T158S-N218S-L257G, T158S-N218S-N243V-K256R-L257G,T158S-N218S-N243V-L257G, T158S-N218S-S248N-K256R-L257G,T158S-N243V-K256R, T158S-N243V-K256R-L257G, T158S-N243V-S248N-K256R,V004A-N109G-A116T-G131H-S248N-K256R-L257G, and Y006H-A116T-G131H-S248N,wherein amino acid positions of the variant are numbered bycorrespondence with the sequence of SEQ ID NO:2. Such variants haveenhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36,and a greater PI value than BPN′, BPN′-v3 and BPN′-v36 in this assay.The invention includes a protease variant having enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2), enhanced proteolytic activitycompared to BPN′, BPN′-v3, and BPN′-v36, a PI value of greater than 1.0to about 5 relative to BPN′-v3, and/or a PI value of greater than 1.0 toabout 5 relative to BPN′-v36 in this assay, the variant comprising anamino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ IDNO:6 and comprising at least one set of amino acid substitutionsselected from said group above, wherein amino acid positions of thevariant are numbered by correspondence with amino acid positions of theSEQ ID NO:2 sequence. Also included are compositions, including, but notlimited to, e.g., cleaning compositions, comprising at least one suchvariant and methods for cleaning utilizing at least one such variant asdescribed in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto about 1.0 relative to BPN′-v36 in an egg microswatch cleaning assayin Detergent Composition 4 at pH 8 and 16° C.:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of BPN′-v36,A015S-A088T-N109G-G131H-T158S-N218S-S248N, A088T,A088T-A116T-G131H-N218S-N243V-S248N, A088T-A116T-G131H-N218S-S248N,A088T-A116T-G131H-N218S-S248N,A088T-A116T-G131H-N218S-S248N-K256R-L257G, A088T-A116T-G131H-N243V,A088T-A116T-G131H-N243V-K256R, A088T-A116T-G131H-N243V-S248N,A088T-A116T-G131H-S248N-K256R,A088T-A116T-G131H-T158S-N218S-N243V-S248N,A088T-A116T-G131H-T158S-N218S-N243V-S248N-K256R,A088T-A116T-G131H-T158S-N218S-N243V-S248N-L257G,A088T-A116T-G131H-T158S-N218S-N243V-S248N-L257G,A088T-A116T-G131H-T158S-N218S-S248N,A088T-A116T-G131H-T158S-N218S-S248N-K256R,A088T-A116T-G131H-T158S-N243V, A088T-A116T-G131H-T158S-N243V-K256R,A088T-A116T-G131H-T158S-N243V-S248N,A088T-A116T-G131H-T158S-N243V-S248N-K256R-L257G,A088T-A116T-G131H-T158S-S248N, A088T-A116T-G131H-T158S-S248N-K256R,A088T-A116T-G131H-T158S-S248N-L257G,A088T-A116T-G131H-V147A-T158S-N218S-N243V-S248N-L257G,A088T-A116T-N218S-I268V, A088T-A116T-N218S-L257G,A088T-A116T-N218S-N243V-N269D, A088T-A116T-N218S-S248N,A088T-A116T-N218S-S248N, A088T-A116T-N218S-S248N-L257G,A088T-A116T-N243V-K256R-L257G, A088T-A116T-N243V-S248N-K256R,A088T-A116T-S248N-K256R, A088T-A116T-T158S-N218S,A088T-A116T-T158S-N218S-N243V-S248N-K256R-L257G,A088T-A116T-T158S-N218S-S248N,A088T-A116T-T158S-N218S-S248N-K256R-L257G,A088T-A116T-T158S-N243V-S248N, A088T-A116T-T158S-N243V-S248N-K256R,A088T-A116T-T158S-N243V-S248N-K256R-L257G,A088T-A116T-T158S-N243V-S248N-L257G, A088T-A116T-T158S-S248N,A088T-A116T-T158S-S248N-K256R-L257G, A088T-A116T-T158S-S248N-L257G,A088T-A116T-V143A-N218S-S248N-K256R, A088T-G131H-A138V-N218S-L257G,A088T-G131H-K141E-N218S-N243V-S248N-L257G, A088T-G131H-K256R,A088T-G131H-N218S-K256R, A088T-G131H-N218S-N243V-K256R-L257G,A088T-G131H-N218S-N243V-S248N, A088T-G131H-N218S-N243V-S248N,A088T-G131H-N218S-N243V-S248N-K256R,A088T-G131H-N218S-N243V-S248N-K256R-L257G, A088T-G131H-N218S-S248N,A088T-G131H-N218S-S248N-K256R, A088T-G131H-N243V-K256R-L257G,A088T-G131H-N243V-S248N, A088T-G131H-N243V-S248N-K256R-L257G,A088T-G131H-S248N, A088T-G131H-S248N-K256R, A088T-G131H-T158S-K256R,A088T-G131H-T158S-L257G, A088T-G131H-T158S-N218S,A088T-G131H-T158S-N218S-N243V-S248N,A088T-G131H-T158S-N218S-N243V-S248N-K256R,A088T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,A088T-G131H-T158S-N218S-N243V-S248N-L257G,A088T-G131H-T158S-N218S-S248N,A088T-G131H-T158S-N218S-S248N-K256R-L257G, A088T-G131H-T158S-N243V,A088T-G131H-T158S-N243V-K256R, A088T-G131H-T158S-N243V-S248N-K256R,A088T-G131H-T158S-N243V-S248N-L257G,A088T-G131H-T158S-S248N-K256R-L257G, A088T-G131H-T158S-S248N-L257G,A088T-I107T-N109G-G131H-N218S-A223G-S248N-K256R, A088T-L257G,A088T-L257G, A088T-N109G-A116T-G131H-A232S-N243V-K256R,A088T-N109G-A116T-G131H-K256R-L257G, A088T-N109G-A116T-G131H-L257G,A088T-N109G-A116T-G131H-N218S-K256R,A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R,A088T-N109G-A116T-G131H-N218S-N243V-S248N-N269D,A088T-N109G-A116T-G131H-N243V-K256R, A088T-N109G-A116T-G131H-S248N,A088T-N109G-A116T-G131H-T158S-L257G,A088T-N109G-A116T-G131H-T158S-N218S-N243F-S248N,A088T-N109G-A116T-G131H-T158S-N218S-N243V,A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,A088T-N109G-A116T-G131H-T158S-N218T-K256R,A088T-N109G-A116T-G131H-T158S-N243V,A088T-N109G-A116T-G131H-T158S-N243V-K256R,A088T-N109G-A116T-G131H-T158S-N243V-S248N,A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R-I268V,A088T-N109G-A116T-G131H-T158S-N243V-S248N-L257G,A088T-N109G-A116T-G131H-T158S-S248N,A088T-N109G-A116T-G131H-T158S-S248N-K256R-L257G,A088T-N109G-A116T-G131H-V149A-N218S-S248N-K256R-L257G,A088T-N109G-A116T-K256R, A088T-N109G-A116T-N218S-K256R,A088T-N109G-A116T-N218S-N243V-S248N-K256R,A088T-N109G-A116T-N218S-N243V-S248N-L257G,A088T-N109G-A116T-N218S-S248N, A088T-N109G-A116T-N218S-S248N-K256R,A088T-N109G-A116T-S248N, A088T-N109G-A116T-T158S-L257G,A088T-N109G-A116T-T158S-N218S, A088T-N109G-A116T-T158S-N243V,A088T-N109G-A116T-T158S-N243V-K256R,A088T-N109G-A116T-T158S-N243V-S248N-K256R,A088T-N109G-G131H-N218S-N243V-S248N,A088T-N109G-G131H-N218S-S248N-K256R,A088T-N109G-G131H-N218S-S248N-K256R-L257G,A088T-N109G-G131H-N218S-S248N-K256R-L257G-Q275R,A088T-N109G-G131H-N218S-S248N-K256R-Q271R, A088T-N109G-G131H-N243V,A088T-N109G-G131H-N243V-K256R, A088T-N109G-G131H-N243V-S248N-K256R,A088T-N109G-G131H-S248N-K256R, A088T-N109G-G131H-S248N-L257G,A088T-N109G-G131H-T158S, A088T-N109G-G131H-T158S,A088T-N109G-G131H-T158S-K256R, A088T-N109G-G131H-T158S-K256R,A088T-N109G-G131H-T158S-N218S-N243V-K256R,A088T-N109G-G131H-T158S-N218S-N243V-K256R,A088T-N109G-G131H-T158S-N218S-N243V-K256R-L257G,A088T-N109G-G131H-T158S-N218S-N243V-S248N-K256R-L257G,A088T-N109G-G131H-T158S-N218S-N243V-S248N-L257G,A088T-N109G-G131H-T158S-N243V-K256R,A088T-N109G-G131H-T158S-N243V-K256R-L257G,A088T-N109G-G131H-T158S-N243V-S248N-K256R-L257G,A088T-N109G-G131H-T158S-S248N, A088T-N109G-G131H-T158S-S248N-L257G,A088T-N109G-G131H-V149A-K256R-L257G,A088T-N109G-G131H-V149L-T158S-K256R-L257G, A088T-N109G-K256R,A088T-N109G-K256R-L257G, A088T-N109G-K256R-L257G, A088T-N109G-L257G,A088T-N109G-N218S-K256R, A088T-N109G-N218S-N243V-K256R,A088T-N109G-N218S-N243V-S248N-K256R,A088T-N109G-N218S-N243V-S248N-K256R, A088T-N109G-N218S-S248N,A088T-N109G-N218S-S248N-L257G, A088T-N109G-N243V-K256R,A088T-N109G-N243V-S248N-L257G-I268V, A088T-N109G-S248N,A088T-N109G-S248N-K256R, A088T-N109G-T158S-K256R-L257G,A088T-N109G-T158S-N218S, A088T-N109G-T158S-N218S-K256R-Q271H,A088T-N109G-T158S-N218S-N243V-K256R,A088T-N109G-T158S-N218S-N243V-K256R-L257G,A088T-N109G-T158S-N218S-N243V-L257G,A088T-N109G-T158S-N218S-N243V-S248N-K256R,A088T-N109G-T158S-N218S-S248N-N269D,A088T-N109G-T158S-N243V-K256R-L257G,A088T-N109G-T158S-N243V-S248N-A274D,A088T-N109G-T158S-N243V-S248N-K256R-L257G-N269D, A088T-N218S-K256R,A088T-N218S-N243V, A088T-N218S-N243V-K256R,A088T-N218S-N243V-K256R-L257G, A088T-N218S-N243V-S248N-K256R,A088T-N218S-N243V-S248N-L257G, A088T-N218S-S248N-L257G,A088T-N243V-S248N, A088T-N243V-S248N-K256R, A088T-N243V-S248N-L257G,A088T-S145T-T158S-S248N, A088T-S248N-K256R-L257G, A088T-S248N-L257G,A088T-T158S, A088T-T158S, A088T-T158S-K256R, A088T-T158S-L257G,A088T-T158S-N218S, A088T-T158S-N218S-K256R,A088T-T158S-N218S-N243V-K256R, A088T-T158S-N218S-N243V-K256R-L257G,A088T-T158S-N218S-N243V-L257G, A088T-T158S-N218S-S248N,A088T-T158S-N218S-S248N, A088T-T158S-N243V, A088T-T158S-N243V-K256R,A088T-T158S-N243V-S248N-L257G, A116T, A116T-G131H-L257G,A116T-G131H-N218S-K256R, A116T-G131H-N218S-N243V-K256R,A116T-G131H-N218S-N243V-K256R-L257G, A116T-G131H-N218S-N243V-S248N,A116T-G131H-N218S-N243V-S248N-K256R, A116T-G131H-N218S-S248N,A116T-G131H-T158S-K256R, A116T-G131H-T158S-N218S-L257G,A116T-G131H-T158S-N218S-N243V-K256R,A116T-G131H-T158S-N218S-N243V-L257G,A116T-G131H-T158S-N218S-N243V-S248N,A116T-G131H-T158S-N218S-S248N-L257G-N269D,A116T-G131H-T158S-N218S-S248N-Q271R, A116T-G131H-T158S-N218T-L257G,A116T-G131H-T158S-N243V-K256R,A116T-G131H-V150A-T158S-N243V-S248N-K256R-L257G,A116T-K141E-N218S-N243V-S248N-K256R-L257G, A116T-L257G,A116T-N218S-N243V-S248N, A116T-N218T-N243V-S248N,A116T-N243V-S248N-L257G, A116T-S248N, A116T-S248N-L257G,A116T-T158S-K256R, A116T-T158S-N218S-L257G,A116T-T158S-N218S-N243V-K256R, A116T-T158S-N218S-N243V-L257G,A116T-T158S-N243V, A116T-T158S-N243V-K256R-L257G,A116T-T158S-N243V-S248N-K256R, A116T-T158S-N243V-S248N-K256R-L257G,A116T-V149I-T158S-N243V-S248N-K256R-Q271H,G024S-G053S-N078S-G097A-N101S, G024S-G053S-N078S-G097A-N101S-A128S,G131H-K256R, G131H-N218S-K256R-L257G, G131H-N218S-N243V-L257G,G131H-N218S-N243V-S248N-K256R, G131H-N218S-S248N-K256R,G131H-N218S-S248N-L257G, G131H-N243V, G131H-N243V-S248N,G131H-N243V-S248N-L257G, G131H-T158S-K256R-L257G, G131H-T158S-N218S,G131H-T158S-N218S-K256R,G131H-T158S-N218S-N240H-N243V-S248N-K256R-L257G,G131H-T158S-N218S-N243V-K256R, G131H-T158S-N218S-N243V-K256R-L257G,G131H-T158S-N218S-N243V-S248N,G131H-T158S-N218S-N243V-S248N-K256R-L257G,G131H-T158S-N218S-S248N-I268V,G131H-T158S-N218S-S248N-K256R-L257G-N269S,G131H-T158S-N243V-K256R-L257G, G131H-T158S-N243V-S248N,G131H-T158S-N243V-S248N-K256R, G131H-T158S-S248N,G131H-T158S-S248N-L257G, G131H-W241L-N243V-S248N-K256R,I107T-G131H-T158S-N243V-S248N-K256R-L257G,I107T-N109G-G131H-N218S-L257G, N109G-A116T-G131H-L257G,N109G-A116T-G131H-N218S-L257G, N109G-A116T-G131H-N218S-N243V,N109G-A116T-G131H-N218S-N243V-S248N-K256R,N109G-A116T-G131H-N218S-W241R-N243V-K256R,N109G-A116T-G131H-N243V-S248N-K256R, N109G-A116T-G131H-S248N-I268V,N109G-A116T-G131H-T158S-N218S,N109G-A116T-G131H-T158S-N218S-S248N-K256R,N109G-A116T-G131H-T158S-N218S-S248N-K256R-L257G,N109G-A116T-G131H-T158S-N218S-S248N-L257G,N109G-A116T-G131H-T158S-N243V-K256R-L257G,N109G-A116T-G131H-T158S-S248N-L257G,N109G-A116T-K141E-T158S-N218S-N243V-L257G, N109G-A116T-K256R,N109G-A116T-N218S-N243V, N109G-A116T-N218S-N243V-S248N,N109G-A116T-N218S-S248N-L257G, N109G-A116T-N243V-S248N-K256R-L257G,N109G-A116T-S248N, N109G-A116T-T158S-N218S-N243V-S248N-K256R,N109G-A116T-T158S-N218S-W241R-N243V, N109G-A116T-T158S-N243V-S248N,N109G-A116T-T158S-S248N-L257G, N109G-G131H-K256R,N109G-G131H-N218S-K256R, N109G-G131H-N218S-N243V,N109G-G131H-N218S-N243V-K256R-L257G,N109G-G131H-N218S-N243V-S248N-K256R, N109G-G131H-N218S-S248N-K256R,N109G-G131H-N243V, N109G-G131H-N243V-S248N,N109G-G131H-N243V-S248N-K256R-L257G, N109G-G131H-S248N,N109G-G131H-T158S-L257G, N109G-G131H-T158S-N218S-N243V-S248N,N109G-G131H-T158S-N218S-N243V-S248N-K256R,N109G-G131H-T158S-N218S-N243V-S248N-K256R-L257G,N109G-G131H-T158S-N243V-L257G, N109G-G131H-T158S-N243V-S248N-K256R,N109G-G131H-T158S-S248N-K256R, N109G-K141E-N218S-S248N-L257G,N109G-N218S-N243V, N109G-N218S-N243V-S248N-K256R,N109G-N218S-S248N-K256R-L257G, N109G-N218S-S248N-L257G,N109G-N243V-S248N-L257G-Q275R, N109G-T158S-I268V, N109G-T158S-N218S,N109G-T158S-N218S-N243V, N109G-T158S-N218S-N243V-S248N,N109G-T158S-N218S-S248N-K256R, N109S-A116T-S248N, N218S-K256R,N218S-N243V-K256R, N218S-N243V-L257G, N218S-N243V-S248N, N218S-S248N,N218S-S248N-K256R, N218S-S248N-K256R-L257G,S003P-N109G-G131H-N218S-N243V-S248N-K256R-L257G, S248N-K256R-L257G,T158S-K256R, T158S-K256R-L257G, T158S-N218S-K256R-L257G,T158S-N218S-L233S-S248N, T158S-N243V, T158S-N243V-S248N-K256R-N269D,T158S-N243V-S248N-L257G, V004L-A116T-N218S-N243V-S248N-L257G, andY006H-N218S-N243V-S248N, wherein amino acid positions of the variant arenumbered by correspondence with the sequence of SEQ ID NO:2. Suchvariants have enhanced proteolytic activity compared to BPN′ (SEQ IDNO:2) and a greater PI value than BPN′ in this assay. The inventionincludes a protease variant having enhanced proteolytic activitycompared to BPN′ (SEQ ID NO:2), a PI value of 1.0 relative to BPN′-v3,and a PI value of 1.0 relative to BPN′-v36 in this assay, the variantcomprising an amino acid sequence having at least 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ IDNO:2 or SEQ ID NO:6 and comprising at least one set of amino acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto or greater than 0.5 and less than 0.9 relative to BPN′-v36 in an eggmicroswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of A088T-A098S-G131H-S248N-K256R-L257G,A088T-A116T-G131H-K256R, A088T-A116T-G131H-N218S-S248N-K256R,A088T-A116T-G131H-N243V-S248N-L257G, A088T-A116T-G131H-S248N,A088T-A116T-G131H-S248N-K256R-L257G, A088T-A116T-G131H-T158S-K256R,A088T-A116T-G131H-T158S-L257G, A088T-A116T-G131H-T158S-N218S-L257G,A088T-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,A088T-A116T-K256R-L257G, A088T-A116T-K256R-L257G,A088T-A116T-N218S-N243V-Q271R, A088T-A116T-N218S-S248N-K256R,A088T-A116T-T158S, A088T-A116T-T158S, A088T-A116T-T158S-K256R,A088T-A116T-T158S-N218S-N243V-L257G,A088T-A116T-T158S-N218S-S248N-K256R, A088T-G131H, A088T-G131H-L257G,A088T-G131H-N218S-N243V-S248N-L257G, A088T-G131H-N243V-K256R,A088T-G131H-N243V-L257G, A088T-G131H-N243V-S248N,A088T-G131H-N243V-S248N-K256R,A088T-G131H-T158S-N218S-I234T-S248N-L257G,A088T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,A088T-G131H-T158S-N243V-K256R-L257G, A088T-G131H-T158S-S248N,A088T-G131H-T158S-S248N-K256R, A088T-G131H-T158S-S248N-K256R-L257G,A088T-G131H-V149L-T158S-N243V-S248N-K256R-L257G,A088T-N109G-A116T-G131H-K141E-N218S,A088T-N109G-A116T-G131H-N218S-N243V-S248N-Q275R,A088T-N109G-A116T-G131H-N218S-S248N,A088T-N109G-A116T-G131H-N243V-S248N-K256R,A088T-N109G-A116T-G131H-S248N-K256R-L257G,A088T-N109G-A116T-G131H-T158S-K256R,A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N,A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R-L257G,A088T-N109G-A116T-G131H-T158S-S248N,A088T-N109G-A116T-G131H-V149A-T158S-N218S-K256R,A088T-N109G-A116T-N218S-S248N-K256R-L257G,A088T-N109G-A116T-N243V-K256R, A088T-N109G-A116T-N243V-S248N-L257G,A088T-N109G-A116T-S248N-L257G,A088T-N109G-A116T-T158S-N212D-N243V-K256R-L257G,A088T-N109G-A137E-T158S-N218S-N243V-S248N-K256R-L257G,A088T-N109G-D140G-N243V,A088T-N109G-G131H-A152S-T158S-N218S-S248N-K256R,A088T-N109G-G131H-D140G-T158S-N243V-S248N-K256R,A088T-N109G-G131H-K256R-L257G, A088T-N109G-G131H-N218S,A088T-N109G-G131H-N218S-N243V, A088T-N109G-G131H-T158S-L257G,A088T-N109G-G131H-T158S-N218S-N243V-S248N-K256R-L257G,A088T-N109G-G131H-T158S-N218S-W241R-S248N-L257G,A088T-N109G-G131H-T158S-S248N-K256R, A088T-N109G-N218S-S248N-K256R,A088T-N109G-N243V-S248N-K256R, A088T-N109G-T158S,A088T-N109G-T158S-N243V-S248N-K256R,A088T-N109G-T158S-N243V-S248N-Q275R, A088T-N109G-T158S-S248N,A088T-N218S-N243V-S248N-K256R-L257G, A088T-N243V-L257G, A088T-S248N,A088T-T158S-K256R, A088T-T158S-N218S-K256R, A088T-T158S-N218S-L257G,A088T-T158S-N218S-L257G, A088T-T158S-N218S-N243V-S248N,A088T-T158S-N218S-N243V-S248N-L257G,A088T-T158S-N218S-Q245K-S248N-K256R,A088T-T158S-N218S-S248N-L257G-Q275K, A088T-T158S-N243V-K256R,A088T-T158S-N243V-K256R-L257G, A088T-T158S-N243V-S248N-K256R,A088T-T158S-S248N, A088T-T158S-S248N-K256R-L257G,A088T-T158S-S248N-L257G, A088T-T158S-S248N-L257G,A098S-G131H-T158S-N218S-N243V-S248N-K256R-L257G,A116T-G131H-K141E-N218S-N243V-S248N-L257G,A116T-G131H-N218S-W241R-N243V-S248N-K256R-L257G, A116T-G131H-N243V,A116T-G131H-T158S-N218S-S248N-K256R,A116T-G131H-V139I-N218S-N243V-S248N, A116T-N218S-S248N-K256R,A116T-T158S-L257G-Q271R, A116T-T158S-N218S-N243V-K256R-L257G,G053S-A088T-N109G-A116T-G131H-T158S-G169S-N218S-S248N-K256R-L257G,G131H-N218S-L257G, G131H-T158S, G131H-T158S-K256R, K256R,L090I-N109G-T158S-N243V, L257G, N109G-A116T-G131H,N109G-A116T-G131H-N243V,N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R,N109G-A116T-S248N-K256R, N109G-A116T-T158S-N218S-K237R-N243V-S248N,N109G-A116T-T158S-S248N, N109G-G131H-T158S,N109G-G131H-T158S-N243V-K256R-L257G, N109G-G131H-T158S-S248N-Q271R,N109G-N218S-S248N-K256R, N109G-N243V-S248N-L257G, N109G-S248N,N109G-T158S-N218S-N243V-L257G, N109G-T158S-N243V-K256R-L257G,N109G-T158S-N243V-S248N-K256R-L257G, N218S-N243V-S248N-K256R-L257G,S003P-N109G-A116T-G131H-T158S-N218S-K256R,S003P-N109G-G131H-T158S-L257G,S105H-W106G-I107L-I108S-N109A-G110A-I111S-E112N-W113G-A114P,T158S-N218S-S248N-K256R, T158S-N243V-S248N, T158S-S248N,T158S-S248N-K256R-L257G, V004A-A088T-A116T-T158S-N218S,V004A-A088T-G131H-N218S-N243V-S248N-L257G,Y006H-N109G-N218S-N243V-S248N, Y104H-A116T-T158S-S248N,A088T-A116T-G131H-N218S-S248N-L257G,A088T-A116T-G131H-T158S-N218S-N243V-L257G,A088T-A116T-G131H-T158S-N243V-K256R-L257G, A088T-A116T-N218S,A088T-G131D-T158S-N243V-S248N, A088T-G131H-N218S-K237R-K256R-L257G,A088T-G131H-N218S-K256R, A088T-K213N-N243V-S248N-K256R,A088T-K256R-L257G, A088T-N109G-A116T-G131H-D140G-S248N-L257G,A088T-N109G-A116T-G131H-L257G,A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R-L257G,A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N,A088T-N109G-A116T-M124I-G131H-T158S-N218S-S248N-L257G,A088T-N109G-A116T-T158S-N218S-N243V-K256R,A088T-N109G-A116T-T158S-N218S-N243V-L257G,A088T-N109G-A116T-T158S-N218S-S248N, A088T-N109G-G131H-K256R-L257G,A088T-N109G-G131H-N218S-N243V-S248N-L257G,A088T-N109G-G131H-N243V-S248N,A088T-N109G-G131H-T158S-L233S-N243V-S248N,A088T-N109G-G131H-T158S-N218S-S248N-K256R, A088T-N109G-L257G,A088T-N109G-N218S-K256R-L257G,A088T-N109G-N218S-S248N-T255K-K256R-L257G,A088T-N109G-S248N-K256R-L257G, A088T-N109G-T158S-N218S-N243V,A088T-N109G-T158S-N218S-N243V-L257G,A088T-N109G-T158S-N218S-N243V-S248N-L257G,A088T-N109G-T158S-N218S-S248N, A088T-S248N-K256R-L257G,A088T-S248N-L257G-I268V, A088T-T158S-N243V-L257G,A088T-T158S-V203I-N218S-K256R-L257G,A088T-Y104H-A116T-G131H-N218S-N243V,A116T-D140G-T158S-N218S-N243V-S248N, A116T-G131H-T158S-K256R-L257G,A116T-G131H-T158S-N243V-S248N, A116T-G157E-T158S-N243V-S248N-K256R,A116T-S248N-K256R, G131H-N218S-S248N, G131H-N243V-K256R,G131H-T158S-N243V-L257G, K256R-L257G,N109G-A116T-G131H-T158S-N218S-N243V-S248N,N109G-A116T-N218S-N243V-L257G,N109G-A116T-N218S-W241R-N243V-S248N-K256R-L257G,N109G-A116T-T158S-N243V, N109G-A116T-T158S-S248N-K256R, N109G-N243V,N109G-T158S-K256R-L257G, N243V,P014L-A015L-L016C-H017T-S018L-Q019K-G020A-Y021T-T022L-G023E,S003P-S248N-L257G, T158S-N218S-N243V-S248N, T158S-S248N-K256R,Y104H-N109G-G131H-N243V-S248N, A088T-A116T-G131H-L257G,A088T-A116T-G131H-N218S-N243V, A088T-A116T-G131H-N218S-S248N-K256R,A088T-A116T-G131H-T158S-N243V-S248N-L257G, A088T-A116T-L257G,A088T-A116T-N218S, A088T-A116T-N218S-N243V-S248N,A088T-A116T-T158S-N218S-S248N, A088T-A116T-T158S-N243V-S248N-K256R,A088T-A138E-N218S-N243V-K256R, A088T-G131H-N218S-N243V-S248N-K256R,A088T-G131H-T158S-S248N-K256R,A088T-N109G-A116T-G131H-N218S-S248N-K256R-L257G,A088T-N109G-A116T-G131H-N243V-S248N-K256R,A088T-N109G-A116T-G131H-T158S-N218S-L257G-I268V,A088T-N109G-A116T-G131H-W241L-S248N-K256R-L257G,A088T-N109G-A116T-N218S-N243V-S248N-K256R,A088T-N109G-A116T-T158S-N243V-K256R-L257G,A088T-N109G-A116T-T158S-N243V-S248N, A088T-N109G-G131H-N218S-L257G,A088T-N109G-G131H-N218S-S248N,A088T-N109G-G131H-T158S-N243V-S248N-L257G,A088T-N109G-N243V-K256R-L257G, A088T-N109G-T158S-S248N-K256R,A088T-N109G-W241R-S248N-K256R, A088T-N218S-S248N-L257G,A088T-T158S-N218S-S248N-K256R, A088T-T158S-N218S-S248N-L257G,A116T-G131H-T158S-N218S-N243V, A116T-N218S-S248N, A116T-N243V-K256R,A116T-T158S, G131H-S248N-K256R, N109G-A116T-G131H-N243V-K256R-L257G,N109G-A116T-I234T-N243V-S248N-K256R-L257G,N109G-A116T-T158S-K256R-L257G, N109G-A116T-T158S-N218S-S248N-K256R,N109G-G131H, N109G-G131H-A137V-T158S-N218S-S248N,N109G-G131H-K141E-L257G, N109G-G131H-T158S-N243V-S248N-L257G,T158S-N218S-N243V-K256R, A088T-A116T-T158S-N243V-K256R-L257G,A088T-N109G-A116T-T158S-K256R-L257G, A088T-N109G-T158S-N218S-S248N,A088T-S248N-L257G,A088T-Y104H-N109G-G131H-A137E-T158S-N218S-N243V-S248N-K256R,G065D-A088T-G131H-N243V-S248N, Y104H-N218S-L257G,A088T-A116T-G131H-V150A-N218S-S248N-L257G,A088T-A116T-T158S-K256R-L257G,A088T-I108T-N109G-G131H-T158S-N218S-S248N-K256R-L257G,A088T-N109G-A116T-T158S-S248N-L257G-Q271P,A088T-N109G-A116T-V148A-N218S-N243V,A088T-Y104H-N109G-A116T-A153S-N218S-N243V-S248N-L257G-N269D, andV004M-A116T-V148A-T158S-N243V-S248N-K256R, wherein amino acid positionsof the variant are numbered by correspondence with the sequence of SEQID NO:2. Such variants have proteolytic activity. The invention includesa protease variant having proteolytic activity and/or a PI value equalto or greater than 0.5 and less than 0.9 relative to BPN′-v36 in thisassay, the variant comprising an amino acid sequence having at least60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least oneset of acid substitutions selected from said group above, wherein aminoacid positions of the variant are numbered by correspondence with aminoacid positions of the SEQ ID NO:2 sequence. Also included arecompositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

Also provided is a subtilisin protease variant having enhancedproteolytic activity compared to BPN′-v36 and/or BPN-′v3 and/or a PIvalue of greater than 1.0 compared to BPN′-v36 in a BMI microswatch oregg microswatch cleaning assay in Detergent Composition 4 at pH 8 and16° C., the variant comprising an amino acid sequence having at least85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ IDNO:2, wherein the variant comprises at least one substitution selectedfrom the group of X001R/T, X002R, X003F/P, X004A/L/M/P, X005S, X006H,X014L, X015L/S, X016C, X017T, X018L, X019K, X020A, X021T, X022L, X023E,X024S, X034S, X053S, X057Q, X065D, X078S, X086L, X088T, X090I, X097A,X098S, X101S, X104H, X105H/P, X106G, X107L/T, X108S/T, X109A/G/S, X110A,X111S, X112N, X113G, X114P/S, X116T, X124I, X128S, X131D/H, X137E/V,X138E/V, X139I, X140G, X141E/R, X143A/F, X144V, X145F/P/T, X146C,X147A/I, X148A, X149A/I/L, X150A, X151S, X152S, X153S, X154A, X155P,X156T, X157E/L, X158M/S, X159E, X160E, X161L, X169S, X182F, X203I,X204F, X207L, X211V, X212D, X213N, X216S, X218S/T, X223G, X231V, X232S,X233S, X234T, X235P, X236F/P, X237N/R, X240H, X241L/R, X243F/V, X245K,X248N, X251K, X255K, X256R, X257G, X260F, X267M, X268V, X269D/S,X271H/K/P/R, X272G/V, X273T, X274D/L/T/V, and X275K/R/S, and optionallyat least one substitution selected from the group of A001R/T, Q002R,S003F/P, V004A/L/M/P, P005S, Y006H, P014L, A015L/S, L016C, H017T, S018L,Q019K, G020A, Y021T, T022L, G023E, G024S, G034S, G053S, P057Q, G065D,N078S, P086L, A088T, L090I, G097A, A098S, N101S, Y104H, S105H/P, W106G,I107L/T, I108S/T, N109A/G/S, G110A, I111S, E112N, W113G, A114P/S, A116T,M124I, A128S, G131D/H, A137E/V, A138E/V, V139I, D140G, K141E/R, V143A/F,A144V, S145F/P/T, G146C, V147A/I, V148A, V149A/I/L, V150A, A151S, A152S,A153S, G154A, N155P, E156T, G157E/L, T158M/S, S159E, G160E, S161L,G169S, S182F, V203I, S204F, S207L, G211V, N212D, K213N, A216S, N218S/T,A223G, A231V, A232S, L233S, I234T, L235P, S236F/P, K237N/R, N240H,W241L/R, N243F/V, Q245K, S248N, E251K, T255K, K256R, L257G, S260F,L267M, I268V, N269D/S, Q271H/K/P/R, A272G/V, A273T, A274D/L/T/V,Q275K/R/S, wherein amino acid positions of the variant are numbered bycorrespondence with positions of the sequence of SEQ ID NO:2. Suchvariants have enhanced proteolytic activity compared to BPN′ (SEQ IDNO:2) BPN′-v3, and BPN′-v36 and a PI value greater than that of BPN′,BPN′-v3, and BPN′-v36 in this assay. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

Example 10 Construction and Cleaning Performance of Additional Variantsof BPN′-v36

The DNA from the site evaluation libraries of the BPN′-v36 (described inExample 7) was further mutagenized by error-prone PCR. These librarieswere amplified with primers P4973 and P4950 (described in Example 7)using Taq DNA polymerase (Promega). Each PCR amplification reactioncontained 30 pmol of each primer, 100 ng of the template DNA (SELs ofthe BPN′-v36) and various amount of MnCl₂. The PCR reaction (20 μL) wasinitially heated at 95° C. for 2.5 min followed by 30 cycles ofdenaturation at 94° C. for 15 sec., annealing at 55° C. for 15 sec. andextension at 72° C. for 2 min. The DNA fragment was gel-purified by theQIAGEN® gel-band purification kit, digested by the BamHI and HindIIIrestriction enzymes and ligated with the pHPLT-BPN′ partial opt vectorthat also was digested with the same restriction enzymes. Ligationmixtures were amplified using rolling circle amplification in anIllustra Templiphi kit according to the manufacturer's recommendation(GE Healthcare) to generate multimeric DNA for transformation intoBacillus subtilis. For this purpose, 1 μl of the ligation mixture wasmixed with 5 μl of the sample buffer, heated to 95° C. for 3 min andcooled on ice. Next, 5 μl of the reaction buffer and 0.2 μl of theenzyme were added to each tube, followed by incubation at 30° C. for 10hours. Products of the rolling circle amplification were diluted 100times and used to transform B. subtilis cells (ΔaprE, ΔnprE,amyE::xylRPxylAcomK-phleo). An aliquot of the transformation mix wasplated on LB plates containing 1.6% skim milk and 10 μg/mL neomycin andincubated overnight at 37° C.

About 500,000 clones were pre-screened on skim milk plates. Very few ofthem formed halos (i.e., indicative of the presence of functionalprotease). Colonies with halos were picked, inoculated in 150 μl of LBmedia containing 10 μg/mL neomycin and sequenced (Quintara). Sequencesof these clones were analyzed by looking for combination of mutations,which occurred in this pool multiple times and might provide performancebenefits. In order to assess the performance of these mutationcombinations, double mutants were created in the BPN′-v36 background byPCR fusion as described below. For this purpose, two or three partiallyoverlapping fragments were amplified by mutagenic primers. Primercombinations used to generate the respective variants are shown in Table10-1 and primer sequences are shown in Table 10-2.

TABLE 10-1 Primers Pairs Used to Amplify Fragments Variant Mutation 1Mutation 2 Fragment 1 Fragment 2 Fragment 3 1 A45S S236Y P4974, P6644,P6647 P6646, P4976 P6645 2 A45S S236G P4974, P6644, P6649 P6648, P4976P6645 3 I115T S183T P4974, P6650, P6655 P6654, P4976 P6651 4 I115V N184YP4974, P6652, P6657 P6656, P4976 P6653 5 I31T S37P P4974, P6658, P4976P6659 6 I31T I35L P4974, P6660, P4976 P6661 7 I31V S38W P4974, P6662,P4976 P6663 8 N25K P129R P4974, P6664, P6667 P6666, P4976 P6665 9 N25KP129K P4974, P6664, P6669 P6668, P4976 P6665 10 P14T S37T P4974, P6670,P6673 P6672, P4976 P6671 11 P5L Q217K P4974, P6678, P6681 P6680, P4976P6679 12 P5L Q217G P4974, P6678, P6683 P6682, P4976 P6679 13 Q10L S37PP4974, P6684, P6675 P6674, P4976 P6685 14 Q10R S37T P4974, P6686, P6673P6672, P4976 P6687 15 S37P T254S P4974, P6674, P6689 P6688, P4976 P667516 N25K S37P P4974, P6664, P6675 P6674, P4976 P6665 17 G24A S37W P4974,P6690, P6677 P6676, P4976 P6691 18 N25K P129R P4974, P6664, P6667 P6666,P4976 P6665 20 S161P S162L P4974, P6694, P4976 P6695 21 S161P T253AP4974, P6692, P6701 P6700, P4976 P6693 22 S161P S260P P4974, P6692,P6703 P6702, P4976 P6693 23 S162L D181H P4974, P6696, P6711 P6710, P4976P6697 24 S162L D181G P4974, P6696, P6713 P6712, P4976 P6697 25 S18FS162L P4974, P6714, P6697 P6696, P4976 P6715 26 S18T S162P P4974, P6716,P6699 P6698, P4976 P6717 27 S18P D120N P4974, P6718, P6727 P6726, P4976P6719 28 S18Y K213R P4974, P6720, P6729 P6728, P4976 P6721 29 S18L Y21SP4974, P6730, P4976 P6731 30 S18T Y21N P4974, P6732, P4976 P6733 31 S9TK141F P4974, P6734, P6737 P6736, P4976 P6635 32 S9T K141R P4974, P6734,P6739 P6738, P4976 P6635 33 Q19L S260N P4974, P6724, P6705 P6704, P4976P6725 34 Q19L S260P P4974, P6724, P6703 P6702, P4976 P6725 35 N61S S260PP4974, P6740, P6703 P6702, P4976 P6741 36 N61D S260I P4974, P6742, P6707P6706, P4976 P6743 37 T253A S260P P4974, P6700, P6703 P6702, P4976 P670138 A134T S260G P4974, P6744, P6709 P6708, P4976 P6745 39 A133V S260NP4974, P6746, P6705 P6704, P4976 P6648

TABLE 10-2 Primer Sequences Used for Generation of Double Mutants ofBPN′-v36 Primer Name SEQ ID NO: P6644 SEQ ID NO: 64 P6645 SEQ ID NO: 65P6646 SEQ ID NO: 66 P6647 SEQ ID NO: 67 P6648 SEQ ID NO: 68 P6649 SEQ IDNO: 69 P6650 SEQ ID NO: 70 P6651 SEQ ID NO: 71 P6652 SEQ ID NO: 72 P6653SEQ ID NO: 73 P6654 SEQ ID NO: 74 P6655 SEQ ID NO: 75 P6656 SEQ ID NO:76 P6657 SEQ ID NO: 77 P6658 SEQ ID NO: 78 P6659 SEQ ID NO: 79 P6660 SEQID NO: 80 P6661 SEQ ID NO: 81 P6662 SEQ ID NO: 82 P6663 SEQ ID NO: 83P6664 SEQ ID NO: 84 P6665 SEQ ID NO: 85 P6666 SEQ ID NO: 86 P6667 SEQ IDNO: 87 P6668 SEQ ID NO: 88 P6669 SEQ ID NO: 89 P6670 SEQ ID NO: 90 P6671SEQ ID NO: 91 P6672 SEQ ID NO: 92 P6673 SEQ ID NO: 93 P6674 SEQ ID NO:94 P6675 SEQ ID NO: 95 P6676 SEQ ID NO: 96 P6677 SEQ ID NO: 97 P6678 SEQID NO: 98 P6679 SEQ ID NO: 99 P6680 SEQ ID NO: 100 P6681 SEQ ID NO: 101P6682 SEQ ID NO: 102 P6683 SEQ ID NO: 103 P6684 SEQ ID NO: 104 P6685 SEQID NO: 105 P6686 SEQ ID NO: 106 P6687 SEQ ID NO: 107 P6688 SEQ ID NO:108 P6689 SEQ ID NO: 109 P6690 SEQ ID NO: 110 P6691 SEQ ID NO: 111 P6692SEQ ID NO: 112 P6693 SEQ ID NO: 113 P6694 SEQ ID NO: 114 P6695 SEQ IDNO: 115 P6696 SEQ ID NO: 116 P6697 SEQ ID NO: 117 P6698 SEQ ID NO: 118P6699 SEQ ID NO: 119 P6700 SEQ ID NO: 120 P6701 SEQ ID NO: 121 P6702 SEQID NO: 122 P6703 SEQ ID NO: 123 P6704 SEQ ID NO: 124 P6705 SEQ ID NO:125 P6706 SEQ ID NO: 126 P6707 SEQ ID NO: 127 P6708 SEQ ID NO: 128 P6709SEQ ID NO: 129 P6710 SEQ ID NO: 130 P6711 SEQ ID NO: 131 P6712 SEQ IDNO: 132 P6713 SEQ ID NO: 133 P6714 SEQ ID NO: 134 P6715 SEQ ID NO: 135P6716 SEQ ID NO: 136 P6717 SEQ ID NO: 137 P6718 SEQ ID NO: 138 P6719 SEQID NO: 139 P6720 SEQ ID NO: 140 P6721 SEQ ID NO: 141 P6722 SEQ ID NO:142 P6723 SEQ ID NO: 143 P6724 SEQ ID NO: 144 P6725 SEQ ID NO: 145 P6726SEQ ID NO: 146 P6727 SEQ ID NO: 147 P6728 SEQ ID NO: 148 P6729 SEQ IDNO: 149 P6730 SEQ ID NO: 150 P6731 SEQ ID NO: 151 P6732 SEQ ID NO: 152P6733 SEQ ID NO: 153 P6734 SEQ ID NO: 154 P6735 SEQ ID NO: 155 P6736 SEQID NO: 156 P6737 SEQ ID NO: 157 P6738 SEQ ID NO: 158 P6739 SEQ ID NO:159 P6740 SEQ ID NO: 160 P6741 SEQ ID NO: 161 P6742 SEQ ID NO: 162 P6743SEQ ID NO: 163 P6744 SEQ ID NO: 164 P6745 SEQ ID NO: 165 P6746 SEQ IDNO: 166 P6747 SEQ ID NO: 167

Each PCR amplification reaction contained 30 pmol of each primer and 100ng of the BPN′-v36 parent template DNA (plasmid pHPLT-BPN′-v36) (seeFIG. 4). Amplifications were carried out using Vent DNA polymerase(NEB). The PCR reaction (20 μL) was initially heated at 95° C. for 2.5min followed by 30 cycles of denaturation at 94° C. for 15 sec.,annealing at 55° C. for 15 sec. and extension at 72° C. for 40 sec.Following amplification, the 5′ and 3′ gene fragments were gel-purifiedby the QIAGEN® gel-band purification kit, mixed (50 ng of eachfragment), mixed and amplified by PCR once again using the primers P4973and P4950 to generate the full-length gene fragment. The PCR conditionswere same as described above, except the extension phase, which wascarried out at 72° C. for 2 min. The full-length DNA fragment wasgel-purified by the QIAGEN® gel-band purification kit, digested by theBamHI and HindIII restriction enzymes and ligated with the pHPLT-BPN′partial opt vector that also was digested with the same restrictionenzymes. Ligation mixtures were amplified using rolling circleamplification in an Illustra Templiphi kit according to themanufacturer's recommendation (GE Healthcare) to generate multimeric DNAfor transformation into Bacillus subtilis. For this purpose, 1 μl of theligation mixture was mixed with 5 μl of the sample buffer, heated to 95°C. for 3 min and cooled on ice. Next, 5 μl of the reaction buffer and0.2 μl of the enzyme were added to each tube, followed by incubation at30° C. for 10 hours. Products of the rolling circle amplification werediluted 100 times and used to transform B. subtilis cells (ΔaprE, ΔnprE,amyE::xylRPxylAcomK-phleo). An aliquot of the transformation mix wasplated on LB plates containing 1.6% skim milk and 10 μg/mL neomycin andincubated overnight at 37° C. Subsequently, the colonies with halos wereinoculated in 150 μl of LB media containing 10 μg/mL neomycin. The nextday, the cultures were either frozen with 15% glycerol or grown in MBDmedium for biochemical analysis as described in Example 2.

The variants were tested for cleaning performance using BMI microswatchassay in Detergent Composition 4 at 16° C. and pH 8, BMI microswatchassay in Detergent Composition 4 at 16° C. and pH 7, and Egg microswatchassay in Detergent Composition 4 at 16° C. and pH 8. Protein content wasdetermined using TCA assay. All assays were performed as described inExample 1 and Performance Indices were calculated relative to BPN′-v36(i.e., BPN′-S24G-S53G-S78N-S101N-G128A-Y217Q).

The following BPN′-v36 variants were determined to have a PI value equalto about 1.0 relative to BPN′-v36 in a BMI microswatch cleaning assay inDetergent Composition 4 at pH 8 and 16° C.:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of BPN′-v36, A133V-S260N, N061S-S260P,P014T-S037T, S009T-K141F, S009T-K141R, S018F-S162L, S018L-Y021S,S018P-D120N, S018T-S162P, S018T-Y021N, S018Y-K213R, S161P-S162L,S161P-S260P, and T253A-S260P, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have enhanced proteolytic activity compared to BPN′ (SEQID NO:2) and a greater PI value than BPN′ in this assay. The inventionincludes a protease variant having enhanced proteolytic activitycompared to BPN′ (SEQ ID NO:2), a PI value of 1.0 relative to BPN′-v3,and a PI value of 1.0 relative to BPN′-v36 in this assay, the variantcomprising an amino acid sequence having at least 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ IDNO:2 or SEQ ID NO:6 and comprising at least one set of amino acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto about 0.9 relative to BPN′-v36 in a BMI microswatch cleaning assay inDetergent Composition 4 at pH 8 and 16° C.:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of A134T-S260G, I115V-N184Y, N025K-S037P,Q010L-S037P, Q019L-S260N, Q019L-S260P, S037P-T254S, and S161P-T253A,wherein amino acid positions of the variant are numbered bycorrespondence with the sequence of SEQ ID NO:2. Such variants haveproteolytic activity. The invention includes a protease variant havingproteolytic activity and/or a PI value of 0.9 relative to BPN′-v36 inthis assay, the variant comprising an amino acid sequence having atleast 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at leastone set of acid substitutions selected from said group above, whereinamino acid positions of the variant are numbered by correspondence withamino acid positions of the SEQ ID NO:2 sequence. Also included arecompositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto or greater than 0.5 and less than 0.9 relative to BPN′-v36 in a BMImicroswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of A045S-S236G, G024A-S037W, I031V-S038W,N061D-S260I, Q010R-S037T, I115T-S183T, N025K-P129K, N025K-P129R,A045S-S236Y, and S162L-D181H, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have proteolytic activity. The invention includes aprotease variant having proteolytic activity and/or a PI value equal toor greater than 0.5 and less than 0.9 relative to BPN′-v36 in thisassay, the variant comprising an amino acid sequence having at least60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least oneset of acid substitutions selected from said group above, wherein aminoacid positions of the variant are numbered by correspondence with aminoacid positions of the SEQ ID NO:2 sequence. Also included arecompositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI valuegreater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from greater than 1.0 to about 10, from greater than1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 7and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence(SEQ ID NO:6) comprising at least one set of amino acid substitutionsselected from the group consisting of S018F-S162L, S018P-D120N,P014T-S037T, S009T-K141R, and S161P-S162L, wherein amino acid positionsof the variant are numbered by correspondence with the sequence of SEQID NO:2. Such variants have enhanced proteolytic activity compared toBPN′, BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3and BPN′-v36 in this assay. The invention includes a protease varianthaving enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2),enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, aPI value of greater than 1.0 to about 5 relative to BPN′-v3, and/or a PIvalue of greater than 1.0 to about 5 relative to BPN′-v36 in this assay,the variant comprising an amino acid sequence having at least 60%, 70%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identityto SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of aminoacid substitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto about 1.0 relative to BPN′-v36 in a BMI microswatch cleaning assay inDetergent Composition 4 at pH 7 and 16° C.:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of BPN′-v36, N061S-S260P, Q010L-S037P,S009T-K141F, S018L-Y021S, S018T-S162P, S018T-Y021N, S018Y-K213R,S037P-T254S, S161P-S260P, S161P-T253A, and T253A-S260P, wherein aminoacid positions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. Such variants have enhanced proteolyticactivity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′in this assay. The invention includes a protease variant having enhancedproteolytic activity compared to BPN′ (SEQ ID NO:2), a PI value of 1.0relative to BPN′-v3, and a PI value of 1.0 relative to BPN′-v36 in thisassay, the variant comprising an amino acid sequence having at least60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least oneset of amino acid substitutions selected from said group above, whereinamino acid positions of the variant are numbered by correspondence withamino acid positions of the SEQ ID NO:2 sequence. Also included arecompositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′ variants were determined to have a PI value of about0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in DetergentComposition 4 at pH 7 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2)comprising at least one set of amino acid substitutions selected fromthe group consisting of A133V-S260N, A134T-S260G, I115T-S183T,I115V-N184Y, N061D-S260I, Q019L-S260N, and Q019L-S260P, whereinpositions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. Such variants have proteolytic activity. Theinvention includes a protease variant having proteolytic activity, a PIvalue of 0.9 relative to BPN′-v3, and/or an enhanced proteolyticactivity compared to BPN′ in this assay, the variant comprising an aminoacid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6and at least one set of amino acid substitutions selected from saidgroup above, wherein amino acid positions of the variant are numbered bycorrespondence with positions of the SEQ ID NO:2 sequence. Also includedare compositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto or greater than 0.5 and less than 0.9 relative to BPN′-v36 in a BMImicroswatch cleaning assay in Detergent Composition 4 at pH 7 and 16°C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of A045S-S236G, G024A-S037W, Q010R-S037T,A045S-S236Y, I031V-S038W, N025K-S037P, S162L-D181H, and N025K-P129R,wherein amino acid positions of the variant are numbered bycorrespondence with the sequence of SEQ ID NO:2. Such variants haveproteolytic activity. The invention includes a protease variant havingproteolytic activity and/or a PI value equal to or greater than 0.5 andless than 0.9 relative to BPN′-v36 in this assay, the variant comprisingan amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 orSEQ ID NO:6 and comprising at least one set of acid substitutionsselected from said group above, wherein amino acid positions of thevariant are numbered by correspondence with amino acid positions of theSEQ ID NO:2 sequence. Also included are compositions, including, but notlimited to, e.g., cleaning compositions, comprising at least one suchvariant and methods for cleaning utilizing at least one such variant asdescribed in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI valuegreater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from greater than 1.0 to about 10, from greater than1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence(SEQ ID NO:6) comprising at least one set of amino acid substitutionsselected from the group consisting of I031V-S038W, P014T-S037T,S018F-S162L, S018P-D120N, and S162L-D181H, wherein amino acid positionsof the variant are numbered by correspondence with the sequence of SEQID NO:2. Such variants have enhanced proteolytic activity compared toBPN′, BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3and BPN′-v36 in this assay. The invention includes a protease varianthaving enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2),enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, aPI value of greater than 1.0 to about 5 relative to BPN′-v3, and/or a PIvalue of greater than 1.0 to about 5 relative to BPN′-v36 in this assay,the variant comprising an amino acid sequence having at least 60%, 70%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identityto SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of aminoacid substitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto about 1.0 relative to BPN′-v36 in an egg microswatch cleaning assayin Detergent Composition 4 at pH 8 and 16° C.:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of BPN′-v36, A133V-S260N, A134T-S260G,G024A-S037W, I1115V-N184Y, N025K-P129K, N025K-P129R, N061D-S260I,Q019L-S260P, S009T-K141F, S009T-K141R, S018L-Y021S, S018T-S162P,S018T-Y021N, S018Y-K213R, S161P-S162L, S161P-T253A, and T253A-S260P,wherein amino acid positions of the variant are numbered bycorrespondence with the sequence of SEQ ID NO:2. Such variants haveenhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and agreater PI value than BPN′ in this assay. The invention includes aprotease variant having enhanced proteolytic activity compared to BPN′(SEQ ID NO:2), a PI value of 1.0 relative to BPN′-v3, and a PI value of1.0 relative to BPN′-v36 in this assay, the variant comprising an aminoacid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6and comprising at least one set of amino acid substitutions selectedfrom said group above, wherein amino acid positions of the variant arenumbered by correspondence with amino acid positions of the SEQ ID NO:2sequence. Also included are compositions, including, but not limited to,e.g., cleaning compositions, comprising at least one such variant andmethods for cleaning utilizing at least one such variant as described ingreater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto or greater than 0.8 and equal to or less than 0.9 relative toBPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acidsequence (SEQ ID NO:6) comprising at least one set of amino acidsubstitutions selected from the group consisting of A045S-S236G,A045S-S236Y, I115T-S183T, N025K-S037P, N061S-S260P, Q010L-S037P,Q010R-S037T, Q019L-S260N, S037P-T254S, and S161P-S260P, wherein aminoacid positions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. Such variants have proteolytic activity. Theinvention includes a protease variant having proteolytic activity and/ora PI value equal to or greater than 0.5 and less than 0.9 relative toBPN′-v36 in this assay, the variant comprising an amino acid sequencehaving at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprisingat least one set of acid substitutions selected from said group above,wherein amino acid positions of the variant are numbered bycorrespondence with amino acid positions of the SEQ ID NO:2 sequence.Also included are compositions, including, but not limited to, e.g.,cleaning compositions, comprising at least one such variant and methodsfor cleaning utilizing at least one such variant as described in greaterdetail elsewhere herein.

Also provided is a subtilisin protease variant having enhancedproteolytic activity compared to BPN′-v36 and/or a PI value of greaterthan 1.0 compared to BPN′-v36 in a BMI or egg microswatch cleaning assayin Detergent Composition 4 at pH 8 and 16° C., the variant comprising anamino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprisesat least one substitution comprising at least one substitution selectedfrom the group of X009T, X010L/R, X014T, X018F/L/P/T/Y, X019L, X021N/S,X024A, X025K, X031V, X037P/T/W, X038W, X045S, X061D/S, X115T/V, X120N,X129K/R, X133V, X134T, X141F/R, X161P, X162L/P, X181H, X183T, X184Y,X213R, X236G/Y, X253A, X254S, and X260G/I/N/P, and optionally at leastone substitution selected from the group of S009T, Q010L/R, P014T,S018F/L/P/T/Y, Q019L, Y021N/S, G024A, N025K, I031V, S037P/T/W, S038W,A045S, N061D/S, I115T/V, D120N, P129K/R, A133V, A134T, K141F/R, S161P,S162L/P, D181H, S183T, N184Y, K213R, S236G/Y, T253A, T254S, andS260G/I/N/P, wherein amino acid positions of the variant are numbered bycorrespondence with positions of the sequence of SEQ ID NO:2. Suchvariants have enhanced proteolytic activity compared to BPN′ (SEQ IDNO:2) BPN′-v3, and BPN′-v36 and a PI value greater than that of BPN′,BPN′-v3, and BPN′-v36 in this assay. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such variant as described in greater detail elsewhere herein.

Example 11

1. Generation of Combinatorial Libraries FS1-FS3

The pHPLT-BPN′-v3 plasmid containing the BPN′ expression cassette servedas template DNA (parent plasmid) for cloning. Three separatecombinatorial libraries (FS1, FS2, and FS3) were synthesized by DNA2.0,and were delivered as individual ligation reactions. A list of librariesand possible substitutions are shown in Table 11-1. The libraries weredesigned to allow the incorporation of either the wild type residues orthe substitutions at each site described in Table 11-1.

For efficient transformation of B. subtilis, the DNA from the ligationreaction was amplified by rolling circle amplification (RCA) using theIllustra Templiphi kit (GE Healthcare). Reactions were performedaccording to the manufacturer's protocol. One microliter of ten-folddiluted amplified DNA was used to transform 50 μL of competent B.subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). Thetransformation mixture was shaken at 37° C. for 1 hour. Ten microliteraliquots of the transformation mixture were plated on skim milk (1.6%)Luria Agar plates supplemented with 10 μg/ml of neomycin (Teknova).

Transformants were picked into microtiter plates containing 125-150 μlLuria broth medium supplemented with 10 μg/ml neomycin. Plates weregrown overnight at 37° C. with 250-300 rpm shaking and 70-80% humidityusing Enzyscreen lids for microtiter plates (Enzyscreen). Between 7 and10 microliters from the overnight culture plate were used to inoculate anew microtiter plate containing 190 μl of MBD medium (a MOPS baseddefined medium) with 10 μg/ml neomycin. MBD medium was preparedessentially as known in the art (see Neidhardt et al., J. Bacteriol.119:736-747 (1974)), except that NH₄Cl, FeSO₄, and CaCl₂ were omittedfrom the base medium, 3 mM K₂HPO₄ was used, and the base medium wassupplemented with 60 mM urea, and 100 ml of a solution made of 210 g/Lglucose, and 350 g/L maltodextrin. The micronutrients were made up as a100× stock solution containing in one liter, 400 mg FeSO₄.7H₂O, 100 mgMnSO₄.H₂O, 100 mg ZnSO₄.7H₂O, 50 mg CuCl₂.2H₂O, 100 mg CoCl₂.6H₂O, 100mg NaMoO₄.2H₂O, 100 mg Na₂B₄O₇.10H₂O, 10 ml of 1 M CaCl₂, and 10 ml of0.5 M sodium citrate. The MBD medium containing microtiter plates weregrown for 60-70 hours at 37° C., 250-300 rpm, and 70-80% humidity usingEnzyscreen lids (Enzyscreen) for determining protein expression. Thenext day, cultures were filtered through a micro-filter plate (0.22 μm;Millipore) and the resulting filtrate was used for biochemical analysis.

TABLE 11-1 Possible Substitutions for Combinatorial Libraries FS1-FS3FS1 FS2 FS3 1 G102A A97G N61E 2 S130P A128G P129E 3 T55P N123G K213L 4V203Y G102A S145D 5 N61P L126V Q275E 6 S101N G100N P40E 7 S53G N62QS159K 8 S78N M124I S24R 9 S87T-A88L- N61P A144K S89G 10 S24G-N25G S130PN240K 11 L75S-N76Y P129S P239R2. Generation of Variants to Improve BPN′ Stability

To improve BPN′ stability, variants were constructed using either parentmolecules pHPLT-BPN′ G97A-G128A-Y217Q-S87D or pHPLT-BPN′G97A-G128A-Y217Q-P40E, both synthesized by Gene Oracle, or parentmolecules pHPLT-BPN′ G97A-G128A-Y217Q-S78N and pHPLT-partial opt FNA (B.amyloliquefaciens subtilisin BPN′-Y217L) synthesized by GeneArt.

The information listed in Tables 11-2 and 11-3 summarizes the parentmolecule used, the mutations added, and the primers used to constructvariants provided herein.

TABLE 11-2 Primer Sequences Used for the Generation of BPN′ StabilityMutants Primer Name SEQ ID NO: p31 169 p32 170 p25 171 p26 172 p33 173p34 174 p29 175 p30 176 p27 177 p28 178 p7 179 p8 180 p21 181 p22 182p11 183 p12 184 p13 185 p14 186 p15 187 p16 188 p17 189 p18 190

TABLE 11-3 Templates Used, Mutations to be Incorporated, and PrimersUsed in the Generation of Variants for Improved Stability Mutation toPrimers Parent Molecules (Templates) Incorporate Used 1 pHPLT-BPN′G97A-G128A-Y217Q- S78N p31, p32 S87D 2 pHPLT-BPN′ G97A-G128A-Y217Q- P40Ep25, p26 S78N 3 pHPLT-BPN′ G97A-G128A-Y217Q- S87D p33, p34 P40E 4pHPLT-BPN′ G97A-G128A-Y217Q- P40E, p25, p33 S78N S87D 5 pHPLT-BPN′G97A-G128A-Y217Q- N76D p29, p30 S87D 6 pHPLT-BPN′ G97A-G128A-Y217Q-N76D, p27, p28 S87D S78N 7 pHPLT-partial opt FNA S78N p7, p8 8pHPLT-partial opt FNA S87D p21, p22 9 pHPLT-partial opt FNA P40E p11,p12 10 pHPLT-partial opt FNA K213N p13, p14 11 pHPLT-partial opt FNAT22V p15, p16 12 pHPLT-partial opt FNA Q206E p17, p18 13 pHPLT-partialopt FNA P40E, p11, p21, S87D, p7 S78N

Bacillus subtilis strains expressing plasmids were streaked onto 1.6%skim milk plates containing 10 ppm neomycin and grown overnight at 37°C. Single colonies from the plates were grown overnight at 37° C. withshaking at 250 rpm in 5 mL Luria broth containing 10 ppm neomycin.Plasmids were isolated using the QIAGEN® Miniprep kit protocol adding 1microliter of Ready Lyse lysozyme (Epicentre) for 15 minutes at 37° C.in buffer P1 to aid in cell lysis. The plasmids were sequenced to ensurecorrect DNA sequences before proceeding. The plasmids were methylatedusing NEB's Dam Methylase Kit in a reaction containing 77.5 μL water+10μL Buffer 10×+0.25 μL SAM+2 μL DAM methylase+10 μL miniprep DNA (˜150ng/μL) at 37° C. overnight. The methylated plasmid DNA was purifiedusing a DNA Clean and Concentrator Kit (Zymo) or with a QIAGEN® PCRpurification kit. Multi-Site QUIKCHANGE® mutagenesis reactions were setup for each of the DNA templates in a reaction mix containing 2.5 μLBuffer 5×+0.75 μL Quik Solution+0.5 μL primer 1 (25 μM)+0.5 μL primer 2(25 μM)+1.5 μL dNTP's+1 μL enzyme blend+16.25 μL H₂O+2 μL DNA. The PCRprogram used was: 95° C. for 1 min; (95° C. for 1 min, 53° C. for 1 min,65° C. for 10 min)×29 cycles; 65° C. for 10 min, 4° C. hold. In allreactions, PCR was performed using a MJ Research PTC-200 Peltier thermalcycler. The parental DNA from the PCR samples was removed by addition of1 μL of DpnI to QUIKCHANGE® mutagenesis kit reactions at 37° C.overnight. To increase the transformation frequency, the DpnI digestedreactions were amplified using rolling circle amplification (RCA) usingthe Illustra TempliPhi kit according to the manufacturer's protocol. B.subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo) weretransformed with 1 μL each of the RCA reaction and the transformed cellswere plated onto LA+1.6% skim milk plates containing 10 ppm neomycin andincubated at 37° C. overnight. Colonies from overnight growth wereselected to perform colony PCR for sequencing using “puReTaq Ready-To-GoPCR Beads” (Amersham). The PCR and sequencing primers used were pHPLT F1(SEQ ID NO:54) and pHPLT seq R1 (SEQ ID NO:55). Clones with appropriatesequences were frozen. The BPN′ variant proteins were produced bygrowing the B. subtilis transformants in 96 well microtiter plates at37° C. for 68 hours in a MOPS based medium containing urea.

3. Generation of BPN′ Variants Derived from Five Different ParentPlasmids

BPN′ variants were constructed using a total of five differenttemplates: BPN′-v3 (G97A-G128A-Y217Q), BPN′-v4 (G97A-N123G-Y217Q), BPN′variant 8, (S87D-G97A-N109D-G128A-S188D-S248R-Y217Q), BPN′ variant 16(S87D-G97A-N109D-G128A-S188D-Y217Q), and BPN′ variant 21(S87R-G97A-N109R-G128A-S188R-Y217Q-S248R) as shown in Table 11-4. Thegeneration of BPN′-v4 and BPN′-v3 are described in PCT App. No.PCT/US09/46066 (WO 09/149144), filed on Jun. 3, 2009, herebyincorporated herein by reference for such description. BPN′ variants 8,16, 21 were synthesized by Gene Oracle and served as parent plasmids tobuild additional variants. All variants were generated using QUIKCHANGE®mutagenesis kits, except two (variants 5 and 33), which were generatedusing fusion PCR as described below. Primers (listed in Table 11-5) forthe generation of variants were synthesized at Integrated DNATechnologies. The mutations introduced (shown in bold) and the primersand template used are shown in Table 11-4.

TABLE 11-4 Mutations Introduced (bold) & Parent Plasmids Used toGenerate BPN′ Variants Variant Variants Constructed Parent PlasmidPrimers Used 1 G97A-N123C-Y217Q G97A-N123G-Y217Q N123C f, N123C r 2N76D-G97A-G128A- G97A-G128A-Y217Q N76D f, N76D r Y217Q 3G97A-N109D-G128A- G97A-G128A-Y217Q N109D f1, N109D r Y217Q 4G97A-G128A-S188D- G97A-G128A-Y217Q S188D f, S188D r Y217Q 5G97A-G128A-S248D- G97A-G128A-Y217Q pHPLT F1, S248D Y217Q forfus, pHPLTR1, S248D revfus 6 G97A-G128A-S188D- G97A-G128A-Y217Q S188D f, S248R f1S248R-Y217Q 7 G97A-N109D-G128A- S87D-G97A-N109D- D87S f, D87S rS188D-S248R-Y217Q G128A-S188D-S248R- Y217Q 8 S87D-G97A-N109D-Synthesized by Gene Synthesized by Gene G128A-S188D-S248R- Oracle OracleY217Q 9 S87R-G97A-G128A- S87D-G97A-N109D- S87R f, S248D f1,S188D-S248D-Y217Q G128A-S188D-Y217Q D109N f 10 S87R-G97A-N109D-S87D-G97A-N109D- R248Sfor, R248Srev G128A-S188D-Y217Q G128A-S188D-S248R-Y217Q 11 G97A-N109D-G128A- G97A-G128A-Y217Q N109D f2, S188R fS188R-Y217Q 12 S87R-G97A-N109D- S87D-G97A-N109D- S87R f, S87R rG128A-S188D-Y217Q- G128A-S188D-S248R- S248R Y217Q 13 G97A-N109D-G128A-G97A-G128A-Y217Q N109D f2, S248R f2 Y217Q-S248R 14 S87D-G97A-G128A-G97A-G128A-Y217Q S87D f, S248R f1 Y217Q-S248R 15 S87D-G97A-N109D-S87D-G97A-N109D- S248D f1, S248D r G128A-S188D-Y217Q- G128A-S188D-Y217QS248D 16 S87D-G97A-N109D- Synthesized by Gene Synthesized by GeneG128A-S188D-Y217Q Oracle Oracle 17 G97A-N109D-G128A- G97A-G128A-Y217QN109D f2, S248D f2 Y217Q-S248D 18 S87R-G97A-G128A- G97A-G128A-Y217Q S87Rf, S87R r Y217Q 19 S87R-G97A-G128A- G97A-G128A-Y217Q S87R f, S248R f1Y217Q-S248R 20 S87R-G97A-G128A- S87R-G97A-N109R- D109N f, D109N rS188R-Y217Q-S248R G128A-S188R- Y217Q-S248R 21 S87R-G97A-N109R-Synthesized by Gene Synthesized by Gene G128A-S188R-Y217Q- Oracle OracleS248R 22 G97A-G102A-G128A- G97A-G128A-Y217Q G102A f, G102A r Y217Q 23G97A-G128A-S130P- G97A-G128A-Y217Q S130P f, S130P r Y217Q 24G97A-S101N-G128A- G97A-G128A-Y217Q S101N f, S101N r Y217Q 25G97A-G100N-G128A- G97A-G128A-Y217Q G100N f, G100N r Y217Q 26N61P-G97A-G128A- G97A-G128A-Y217Q N61P f, N61P r Y217Q 27G97A-G128A-A187D- G97A-G128A-Y217Q A187D f, A187D r Y217Q 28G97A-G128A-F189D- G97A-G128A-Y217Q f189D f, f189D r Y217Q 29G97A-G128A-A137V- G97A-G128A-Y217Q A137V f, A137V r Y217Q 30S63T-G97A-G128A- G97A-G128A-Y217Q S63T f, S63T r Y217Q 31G97A-Q103N-G128A- G97A-G128A-Y217Q Q103N f, Q103N r Y217Q 32N62D-G97A-G128A- G97A-G128A-Y217Q N62D f, N62D r Y217Q 33G97A-G100E-G128A- G97A-G128A-Y217Q G100E Fsfor, pHPLT Y217Q F1, G100EFsrev, pHPLT R1Generation of BPN′ Variants Via QUIKCHANGE® Mutagenesis

Bacillus subtilis strains containing plasmids expressing BPN′-v3,BPN′-v4, BPN′ variant 8, BPN′ variant 16, and BPN′ variant 21 werestreaked onto 1.6% skim milk plates containing 10 ppm neomycin and grownovernight at 37° C. Single colonies from the plates were grown overnightat 37° C. with shaking at 250 rpm in 5 mL Luria broth containing 10 ppmneomycin. Plasmids expressing BPN′-v3, BPN′-v4, BPN′ variant 8, BPN′variant 16, and BPN′ variant 21 were isolated using QIAGEN® Miniprep kitprotocol except following cell lysis, 1 microliter of Ready Lyselysozyme was added and incubated for 15 minutes at 37° C. The plasmidswere methylated using NEB's Dam Methylase Kit in a reaction containing77.75 μL H₂O+10 μL Buffer 10×+0.25 μL SAM+2 μL DAM methylase+10 μLminiprep DNA at 37° C. overnight. The methylated DNA was purified usingthe QIAGEN® PCR purification kit. Variants 14, 18, and 19 listed inTable 11-4 were generated using QUIKCHANGE LIGHTNING™ MultiSite-Directed Mutagenesis kits (Stratagene) in a reaction mix containing2.5 μL Buffer 5×+0.75 μL Quik Solution+0.5 μL primer 1 (25 μM)+0.5 μLprimer 2 (25 μM)+1.5 μL dNTP's+1 μL enzyme blend+16.25 μL H₂O+2 μL DNA.The PCR program used was as follows: 95° C. for 2 min; (95° C. for 20sec, 55° C. for 30 sec, 64° C. for 2 min 30 sec)×29 cycles; 64° C. for 5min, 4° C. hold.

The remaining variants were created using QUIKCHANGE® MultiSite-Directed Mutagenesis kits in a reaction mix containing 2.5 μLBuffer 5×+0.75 μL Quik Solution+0.5 μL primer 1 (25 μM)+0.5 μL primer 2(25 μM)+1.5 μL dNTP's+1 μL enzyme blend+16.25 μL H₂O+2 μL DNA. The PCRprogram used was as follows: 95° C. for 1 min; (95° C. for 1 min, 53° C.for 1 min, 65° C. for 10 min)×29 cycles; 65° C. for 10 min, 4° C. hold.In all reactions, PCR was performed using a MJ Research PTC-200 Peltierthermal cycler. The primers used for the Quik-Change reactions areprovided in Table 11-5. The primers are shown in primer sequence (5′ to3′).

TABLE 11-5 Primers Used for Quik-Change Reactions Primer Name SEQ ID NO:N76D f 191 N76D r 192 S87D f 193 S87D r 194 G102A f 195 G102A r 196S130P f 197 S130P r 198 S101N f 199 S101N r 200 G100N f 201 G100N r 202N61P f 203 N61P r 204 A187D f 205 A187D r 206 F189D f 207 F189D r 208A137V f 209 A137V r 210 S63T f 211 S63T r 212 Q103N f 213 Q103N r 214N62D f 215 N62D r 216 N109D f1 217 N109D r 218 S188D f 219 S188D r 220S248R f1 221 S87R f 222 S87R r 223 S248D f1 224 S248D r 225 D87S f 226D87S r 227 D109N f 228 D109N r 229 N109D f2 230 S248R f2 231 S248D f2232 S188R f 233 QC FUSION_For1 234 QC FUSION_Rev1 235 S248D forfus 236S248D revfus 237 R248Sfor 238 R248S rev 239 G100E_Fsfor 240 G100E_Fsrev241 N123C f 242 N123C r 243 pHpLT R1 244

The parental DNA from the PCR samples was removed by addition of 1 μL ofDpnI to Quik-Change reactions at 37° C. overnight. One micro-liter ofthe DpnI-digested reactions were amplified using rolling circleamplification (RCA) using the Illustra TempliPhi kit according to themanufacturer's protocol. B. subtilis cells (ΔaprE, ΔnprE,amyE::xylRPxylAcomK-phleo) were transformed with 1 μL each of the RCAreaction and the transformed cells were plated onto LA+1.6% skim milkplates containing 10 ppm neomycin and incubated at 37° C. overnight.Colonies from overnight growth were selected to perform colony PCR using“puReTaq Ready-To-Go PCR Beads” (Amersham). The PCR primers used werepHPLT F1 (SEQ ID NO:54) and pHPLT seq R1 (SEQ ID NO:55). Clones withappropriate sequences were frozen. BPN′ variants were expressed bygrowing the B. subtilis transformants in 96 well microtiter plates at37° C. for 68 hours in a MOPS based medium containing urea.

Generation of BPN′ Variants Via Fusion PCR

Variants 5 and 33 were generated using Fusion PCR, with fragmentsamplified from template pHPLT-BPN′-v3. The PCR primers used to generatethese variants are included in Table 11-5. For Variant 5, a 5′ fragmentof the BPN′ gene was amplified using forward primer pHPLT F1(SEQ IDNO:54), and reverse primer S248D revfus. The 3′ fragment of the BPN′gene was amplified using the forward primer S248D forfus containing themutation of interest and the reverse primer pHPLT RE The two productscontained 20 bp of overlapping sequence, and were fused by combining 1μL of each fragment and fusion primers QC FUSION_For1 and QC FUSION_Rev1in a final PCR reaction. All PCR reactions were performed using standardconditions of the Herculase II PCR Kit (Stratagene). The PCR mixcontained 1 μL DNA polymerase, 1 μL plasmid DNA (or fragment DNA forfusion), 0.5 μL dNTP's, 1.25 μL 25 μM forward primer, 1.25 μL 25 μMreverse primer, 10 μL Buffer 5×, 35 μL H₂O and the PCR program used wasas follows: 95° C. for 2 min, (95° C. for 30 sec, 55° C. for 30 sec, 72°C. for “X” sec) for 29 cycles, 72° C. for 1 min, 4° C. hold (the “X” is15 seconds per 1 kB of DNA to amplify).

For Variant 33, a 5′ fragment of the BPN′ gene was amplified using thetemplate pHPLT-BPN′-v3 and primers pHPLT F1 (SEQ ID NO:54), andG100E_Fsrev. The 3′ fragment that contained the variant was amplifiedusing primers G100E_Fsfor and pHPLT RE The two products contained 20 bpof overlapping sequence, and were fused by combining 1 μl of eachfragment and fusion primers QC FUSION_For1 and QC FUSION_Rev1 in a finalPCR reaction. The PCR conditions were the same as listed above.

The two fusion products were purified using a QIAGEN® PCR purificationcolumn with conditions provided by the manufacturer, and digestedovernight using Bgl I and HindIII enzymes. The plasmid pHPLT-BPN′partial opt was digested using the same enzymes and the vector band wasgel extracted and purified over a QIAGEN® gel purification column usingthe manufacturer's recommendations. The restriction enzyme mixcontained: 10 μL purified DNA, 5 μL Roche Buffer B, 0.5 μL HindIII, 0.5μL Bgl I, 34 μL H₂O and the reactions were carried out at 37° C. for 8hours followed by 65° C. for 20 min. The digest was purified using aQIAGEN® PCR purification column and ligated to the cut vector backboneovernight at 16° C. using the Mighty Mix Ligase kit (Tekara). Followingincubation, 1 μL of the ligation mix was amplified using the IllustraTempliPhi kit.

For the amplification reaction, 1 μL of the ligation reaction mix wasmixed with 5 μL of sample buffer from the TempliPhi kit and heated for 3minutes at 95° C. to denature the DNA. The reaction was placed on ice tocool for 2 minutes and then spun down briefly. Five microliters ofreaction buffer and 0.2 μL of phi29 polymerase from the TempliPhi kitwere added, and the reactions were incubated at 30° C. in an MJ ResearchPCR machine for 4 hours. The phi29 enzyme was heat inactivated in thereactions by incubation at 65° C. for 10 min in the PCR machine.Bacillus subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo) weretransformed using 1 μL of the reaction mix and the transformants weregrown overnight at 37° C. on 1.6% skim milk plates containing 10 ppmneomycin. Transformants were selected to perform colony PCR andsequencing using “puReTaq Ready-To-Go PCR Beads” (Amersham) and primerspHPLT F1 (SEQ ID NO:54) and pHPLT seqR1 (SEQ ID NO:55).

4. Generation of BPN′ Variants from Libraries RCL4-RCL7

RCL4 Library

“RCL4” refers to a group of site saturation libraries created by PCRfusion that simultaneously randomize three contiguous codons in theBPN′-v3-encoding (BPN′-G97A-G128A-Y217Q) gene. The amino acid positionscorresponding to the three mutated codons in each library are providedin Table 11-6. Two partially overlapping, complementary mutagenicprimers, each containing three degenerate codons were used to introducemutations within each library as shown in Table 11-6 below. Only thefirst two nucleotides of each degenerate codon (NNX, N=A, C, T, or G andX is unchanged nucleotide) of interest were mutated in each primer(Table 11-6).

To create each library, two PCR reactions were carried out using eitherthe common 3′ gene-flanking primer (P4976, CCTCTCGGTTATGAGTTAGTTC; (SEQID NO:61)) and mutagenic primer, or the common 5′gene-flanking primer(P4974, GCCTCACATTTGTGCCACCTA; (SEQ ID NO:60)) and mutagenic primer asshown for each library in Table 11-6. These PCR reactions generated twoPCR fragments, one encoding the 5′ half of the mutant BPN′-v3 gene (5′gene fragment) and the other encoding the 3′ half of the mutant BPN′-v3gene (3′ gene fragment). Each PCR amplification reaction contained 30pmol of each primer and 100 ng of the BPN′-v3 parent template DNA(plasmid pHPLT-BPN′-v3) (see FIG. 1). Amplifications were carried outusing Vent DNA polymerase (NEB). The PCR reaction (20 μL) was initiallyheated at 95° C. for 2.5 min followed by 30 cycles of denaturation at94° C. for 15 sec., annealing at 55° C. for 15 sec. and extension at 72°C. for 40 sec. Following amplification, the 5′ and 3′ gene fragmentswere gel-purified by the QIAGEN® gel-band purification kit, mixed (50 ngof each fragment) and amplified by PCR once again using the primersP4973 (AAAGGATCCTAATCGGCGCTTTTC; SEQ ID NO:62) and P4950(CTTGTCTCCAAGCTTAAAATAAAA; SEQ ID NO:63) to generate the full-lengthgene fragment. The PCR conditions were same as described above, exceptthe extension phase, which was carried out at 72° C. for 2 min. Thefull-length DNA fragment was gel-purified by the QIAGEN® gel-bandpurification kit, digested by the BamHI and HindIII restriction enzymesand ligated with the pHPLT-BPN′ partial opt vector that also wasdigested with the same restriction enzymes. Ligation mixtures wereamplified using rolling circle amplification in an Illustra Templiphikit according to the manufacturer's recommendation (GE Healthcare) togenerate multimeric DNA for transformation into Bacillus subtilis. Forthis purpose, 1 μl of the ligation mixture was mixed with 5 μl of thesample buffer, heated to 95° C. for 3 min and cooled on ice. Next, 5 μlof the reaction buffer and 0.2 μl of the enzyme were added to each tube,followed by incubation at 30° C. for 10 hours. Products of the rollingcircle amplification were diluted 100 times and used to transform B.subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). An aliquot ofthe transformation mix was plated on LB plates containing 1.6% skim milkand 10 μg/mL neomycin and incubated overnight at 37° C. Subsequently,the colonies with halos were inoculated in 120 μl of LB media containing10 μg/mL neomycin.

TABLE 11-6 List of Primers Used to Create RCL4 Libraries Mutatedresidues Common in flanking Mutagenic Library BPN′- primer primer # v3Gene fragment names names SEQ ID NO: 1 5-7 3′ P4976 P5119 245 5′ P4974P5120 246 2 11-13 3′ P4976 P5121 247 5′ P4974 P5122 248 3 20-22 3′ P4976P5123 249 5′ P4974 P5124 250 4 21-23 3′ P4976 P5125 251 5′ P4974 P5126252 5 22-24 3′ P4976 P5127 253 5′ P4974 P5128 254 6 23-25 3′ P4976 P5129255 5′ P4974 P5130 256 7 24-26 3′ P4976 P5131 257 5′ P4974 P5132 258 825-27 3′ P4976 P5133 259 5′ P4974 P5134 260 9 26-28 3′ P4976 P5135 2615′ P4974 P5136 262 10 27-29 3′ P4976 P5137 263 5′ P4974 P5138 264 1128-30 3′ P4976 P5139 265 5′ P4974 P5140 266 12 29-31 3′ P4976 P5141 2675′ P4974 P5142 268 13 30-32 3′ P4976 P5143 269 5′ P4974 P5144 270 1431-33 3′ P4976 P5145 271 5′ P4974 P5146 272 15 32-34 3′ P4976 P5147 2735′ P4974 P5148 274 16 33-35 3′ P4976 P5149 275 5′ P4974 P5150 276 1734-36 3′ P4976 P5151 277 5′ P4974 P5152 278 18 35-37 3′ P4976 P5153 2795′ P4974 P5154 280 19 36-38 3′ P4976 P5155 281 5′ P4974 P5156 282 2037-39 3′ P4976 P5157 283 5′ P4974 P5158 284 21 38-40 3′ P4976 P5159 2855′ P4974 P5160 286 22 41-43 3′ P4976 P5161 287 5′ P4974 P5162 288 2343-45 3′ P4976 P5163 289 5′ P4974 P5164 290 24 44-46 3′ P4976 P5165 2915′ P4974 P5166 292 25 51-53 3′ P4976 P5167 293 5′ P4974 P5168 294 2652-54 3′ P4976 P5169 295 5′ P4974 P5170 296 27 53-55 3′ P4976 P5171 2975′ P4974 P5172 298 28 54-56 3′ P4976 P5173 299 5′ P4974 P5174 300 2955-57 3′ P4976 P5175 301 5′ P4974 P5176 302 30 56-58 3′ P4976 P5177 3035′ P4974 P5178 304 31 57-59 3′ P4976 P5179 305 5′ P4974 P5180 306 3258-60 3′ P4976 P5181 307 5′ P4974 P5182 308 33 59-61 3′ P4976 P5183 3095′ P4974 P5184 310 34 60-62 3′ P4976 P5185 311 5′ P4974 P5186 312 3561-63 3′ P4976 P5187 313 5′ P4974 P5188 314 36 62-64 3′ P4976 P5189 3155′ P4974 P5190 316 37 67-69 3′ P4976 P5191 317 5′ P4974 P5192 318 3868-70 3′ P4976 P5193 319 5′ P4974 P5194 320 39 71-73 3′ P4976 P5195 3215′ P4974 P5196 322 40 74-76 3′ P4976 P5197 323 5′ P4974 P5198 324 4177-79 3′ P4976 P5199 325 5′ P4974 P5200 326 42 81-83 3′ P4976 P5201 3275′ P4974 P5202 328 43 83-85 3′ P4976 P5203 329 5′ P4974 P5204 330 4484-86 3′ P4976 P5205 331 5′ P4974 P5206 332 45 85-87 3′ P4976 P5207 3335′ P4974 P5208 334 46 86-88 3′ P4976 P5209 335 5′ P4974 P5210 336 4787-89 3′ P4976 P5211 337 5′ P4974 P5212 338 48 88-90 3′ P4976 P5213 3395′ P4974 P5214 340 49 89-91 3′ P4976 P5215 341 5′ P4974 P5216 342 5090-92 3′ P4976 P5217 343 5′ P4974 P5218 344 51 91-93 3′ P4976 P5219 3455′ P4974 P5220 346 52 92-94 3′ P4976 P5221 347 5′ P4974 P5222 348 5393-95 3′ P4976 P5223 349 5′ P4974 P5224 350 54 94-96 3′ P4976 P5225 3515′ P4974 P5226 352 55 95-97 3′ P4976 P5227 353 5′ P4974 P5228 354 5696-98 3′ P4976 P5229 355 5′ P4974 P5230 356 57 97-99 3′ P4976 P5231 3575′ P4974 P5232 358 58  98-100 3′ P4976 P5233 359 5′ P4974 P5234 360 59 99-101 3′ P4976 P5235 361 5′ P4974 P5236 362 60 100-102 3′ P4976 P5237363 5′ P4974 P5238 364 61 101-103 3′ P4976 P5239 365 5′ P4974 P5240 36662 102-104 3′ P4976 P5241 367 5′ P4974 P5242 368 63 103-105 3′ P4976P5243 369 5′ P4974 P5244 370 64 104-106 3′ P4976 P5245 371 5′ P4974P5246 372 65 105-107 3′ P4976 P5247 373 5′ P4974 P5248 374 66 106-108 3′P4976 P5249 375 5′ P4974 P5250 376 67 107-109 3′ P4976 P5251 377 5′P4974 P5252 378 68 108-110 3′ P4976 P5253 379 5′ P4974 P5254 380 69109-111 3′ P4976 P5255 381 5′ P4974 P5256 382 70 110-112 3′ P4976 P5257383 5′ P4974 P5258 384 71 111-113 3′ P4976 P5259 385 5′ P4974 P5260 38672 112-114 3′ P4976 P5261 387 5′ P4974 P5262 388 73 113-115 3′ P4976P5263 389 5′ P4974 P5264 390 74 114-116 3′ P4976 P5265 391 5′ P4974P5266 392 75 115-117 3′ P4976 P5267 393 5′ P4974 P5268 394 76 116-118 3′P4976 P5269 395 5′ P4974 P5270 396 77 117-119 3′ P4976 P5271 397 5′P4974 P5272 398 78 118-120 3′ P4976 P5273 399 5′ P4974 P5274 400 79119-121 3′ P4976 P5275 401 5′ P4974 P5276 402 80 120-122 3′ P4976 P5277403 5′ P4974 P5278 404 81 121-123 3′ P4976 P5279 405 5′ P4974 P5280 40682 122-124 3′ P4976 P5281 407 5′ P4974 P5282 408 83 123-125 3′ P4976P5283 409 5′ P4974 P5284 410 84 124-126 3′ P4976 P5285 411 5′ P4974P5286 412 85 125-127 3′ P4976 P5287 413 5′ P4974 P5288 414 86 126-128 3′P4976 P5289 415 5′ P4974 P5290 416 87 129-131 3′ P4976 P5291 417 5′P4974 P5292 418 88 132-134 3′ P4976 P5293 419 5′ P4974 P5294 420 89133-135 3′ P4976 P5295 421 5′ P4974 P5296 422 90 134-136 3′ P4976 P5297423 5′ P4974 P5298 424 91 135-137 3′ P4976 P5299 425 5′ P4974 P5300 42692 136-138 3′ P4976 P5301 427 5′ P4974 P5302 428 93 137-139 3′ P4976P5303 429 5′ P4974 P5304 430 94 138-140 3′ P4976 P5305 431 5′ P4974P5306 432 95 139-141 3′ P4976 P5307 433 5′ P4974 P5308 434 96 140-142 3′P4976 P5309 435 5′ P4974 P5310 436 97 141-143 3′ P4976 P5311 437 5′P4974 P5312 438 98 142-144 3′ P4976 P5313 439 5′ P4974 P5314 440 99143-145 3′ P4976 P5315 441 5′ P4974 P5316 442 100 144-146 3′ P4976 P5317443 5′ P4974 P5318 444 101 145-147 3′ P4976 P5319 445 5′ P4974 P5320 446102 146-148 3′ P4976 P5321 447 5′ P4974 P5322 448 103 147-149 3′ P4976P5323 449 5′ P4974 P5324 450 104 148-150 3′ P4976 P5325 451 5′ P4974P5326 452 105 149-151 3′ P4976 P5327 453 5′ P4974 P5328 454 106 150-1523′ P4976 P5329 455 5′ P4974 P5330 456 107 151-153 3′ P4976 P5331 457 5′P4974 P5332 458 108 158-160 3′ P4976 P5333 459 5′ P4974 P5334 460 109159-161 3′ P4976 P5335 461 5′ P4974 P5336 462 110 160-162 3′ P4976 P5337463 5′ P4974 P5338 464 111 163-165 3′ P4976 P5339 465 5′ P4974 P5340 466112 164-166 3′ P4976 P5341 467 5′ P4974 P5342 468 113 167-169 3′ P4976P5343 469 5′ P4974 P5344 470 114 168-170 3′ P4976 P5345 471 5′ P4974P5346 472 115 169-171 3′ P4976 P5347 473 5′ P4974 P5348 474 116 170-1723′ P4976 P5349 475 5′ P4974 P5350 476 117 171-173 3′ P4976 P5351 477 5′P4974 P5352 478 118 172-174 3′ P4976 P5353 479 5′ P4974 P5354 480 119182-184 3′ P4976 P5355 481 5′ P4974 P5356 482 120 183-185 3′ P4976 P5357483 5′ P4974 P5358 484 121 184-186 3′ P4976 P5359 485 5′ P4974 P5360 486122 185-187 3′ P4976 P5361 487 5′ P4974 P5362 488 123 186-188 3′ P4976P5363 489 5′ P4974 P5364 490 124 192-194 3′ P4976 P5365 491 5′ P4974P5366 492 125 194-196 3′ P4976 P5367 493 5′ P4974 P5368 494 126 195-1973′ P4976 P5369 495 5′ P4974 P5370 496 127 196-198 3′ P4976 P5371 497 5′P4974 P5372 498 128 197-199 3′ P4976 P5373 499 5′ P4974 P5374 500 129198-200 3′ P4976 P5375 501 5′ P4974 P5376 502 130 203-205 3′ P4976 P5377503 5′ P4974 P5378 504 131 210-212 3′ P4976 P5379 505 5′ P4974 P5380 506132 211-213 3′ P4976 P5381 507 5′ P4974 P5382 508 133 216-218 3′ P4976P5383 509 5′ P4974 P5384 510 134 217-219 3′ P4976 P5385 511 5′ P4974P5386 512 135 218-220 3′ P4976 P5387 513 5′ P4974 P5388 514 136 219-2213′ P4976 P5389 515 5′ P4974 P5390 516 137 230-232 3′ P4976 P5391 517 5′P4974 P5392 518 138 231-233 3′ P4976 P5393 519 5′ P4974 P5394 520 139232-234 3′ P4976 P5395 521 5′ P4974 P5396 522 140 238-240 3′ P4976 P5397523 5′ P4974 P5398 524 141 240-242 3′ P4976 P5399 525 5′ P4974 P5400 526142 246-248 3′ P4976 P5401 527 5′ P4974 P5402 528 143 255-257 3′ P4976P5403 529 5′ P4974 P5404 530 144 258-260 3′ P4976 P5405 531 5′ P4974P5406 532 145 265-267 3′ P4976 P5407 533 5′ P4974 P5408 534RCL5 Variants

“RCL5” refers to a set of combinatorial variants created by PCR fusionusing several BPN′ mutants as parent (template) molecules. The mutationsintroduced in each parent plasmid are shown in Table 11-7 and themutagenic primers used to create the mutants are indicated in Table11-8.

TABLE 11-7 List of Parent Plasmids and Mutations Introduced in the RCL5Variants Combi- natorial Mutations Variant # Parent Plasmids Introduced1 G97A-G128A-Y217Q-T22N-S24A N61P-N62S 2 G97A-G128A-Y217Q-T22N-S24A T55P3 G97A-G128A-Y217Q-T22N-S24A N61P-S63H 4 G97A-G128A-Y217Q-T22N-S24AQ59S-N61P 5 G97A-G128A-Y217Q-T22N-S24A L75S-N76Y 6G97A-G128A-Y217Q-T22N-S24A P86S-S87G-A88V 7 G97A-G128A-Y217Q-T22N-S24AS87G-A88V-S89A 8 G97A-G128A-Y217Q-T22N-S24A S87T-A88L-S89G 9G97A-G128A-Y217Q-T22N-S24A P129Q-S130G-G131S 10G97A-G128A-Y217Q-T22N-S24A V203Y 11 G97A-G128A-Y217Q-T22N-S24AG211R-N212S-K213V 12 G97A-G128A-Y217Q-S24G-N25G N61P-N62S 13G97A-G128A-Y217Q-S24G-N25G T55P 14 G97A-G128A-Y217Q-S24G-N25G N61P-S63H15 G97A-G128A-Y217Q-S24G-N25G Q59S-N61P 16 G97A-G128A-Y217Q-S24G-N25GL75S-N76Y 17 G97A-G128A-Y217Q-S24G-N25G P86S-S87G-A88V 18G97A-G128A-Y217Q-S24G-N25G S87G-A88V-S89A 19 G97A-G128A-Y217Q-S24G-N25GS87T-A88L-S89G 20 G97A-G128A-Y217Q-S24G-N25G P129Q-S130G-G131S 21G97A-G128A-Y217Q-S24G-N25G V203Y 22 G97A-G128A-Y217Q-S24G-N25GG211R-N212S-K213V 23 G97A-G128A-Y217Q-S24R N61P-N62S 24G97A-G128A-Y217Q-S24R T55P 25 G97A-G128A-Y217Q-S24R N61P-S63H 26G97A-G128A-Y217Q-S24R Q59S-N61P 27 G97A-G128A-Y217Q-S24R L75S-N76Y 28G97A-G128A-Y217Q-S24R P86S-S87G-A88V 29 G97A-G128A-Y217Q-S24RS87G-A88V-S89A 30 G97A-G128A-Y217Q-S24R S87T-A88L-S89G 31G97A-G128A-Y217Q-S24R P129Q-S130G-G131S 32 G97A-G128A-Y217Q-S24R V203Y33 G97A-G128A-Y217Q-S24R G211R-N212S-K213V 34G97A-G128A-Y217Q-G23A-S24G-N25G N61P-N62S 35G97A-G128A-Y217Q-G23A-S24G-N25G T55P 36 G97A-G128A-Y217Q-G23A-S24G-N25GN61P-S63H 37 G97A-G128A-Y217Q-G23A-S24G-N25G Q59S-N61P 38G97A-G128A-Y217Q-G23A-S24G-N25G L75S-N76Y 39G97A-G128A-Y217Q-G23A-S24G-N25G P86S-S87G-A88V 40G97A-G128A-Y217Q-G23A-S24G-N25G S87G-A88V-S89A 41G97A-G128A-Y217Q-G23A-S24G-N25G S87T-A88L-S89G 42G97A-G128A-Y217Q-G23A-S24G-N25G P129Q-S130G-G131S 43G97A-G128A-Y217Q-G23A-S24G-N25G V203Y 44 G97A-G128A-Y217Q-G23A-S24G-N25GG211R-N212S-K213V 45 G97A-G128A-Y217Q-N61P-N62S L75S-N76Y 46G97A-G128A-Y217Q-N61P-N62S P86S-S87G-A88V 47 G97A-G128A-Y217Q-N61P-N62SS87G-A88V-S89A 48 G97A-G128A-Y217Q-N61P-N62S S87T-A88L-S89G 49G97A-G128A-Y217Q-N61P-N62S P129Q-S130G-G131S 50G97A-G128A-Y217Q-N61P-N62S V203Y 51 G97A-G128A-Y217Q-N61P-N62SG211R-N212S-K213V 52 G97A-G128A-Y217Q-T55P L75S-N76Y 53G97A-G128A-Y217Q-T55P P86S-S87G-A88V 54 G97A-G128A-Y217Q-T55PS87G-A88V-S89A 55 G97A-G128A-Y217Q-T55P S87T-A88L-S89G 56G97A-G128A-Y217Q-T55P P129Q-S130G-G131S 57 G97A-G128A-Y217Q-T55P V203Y58 G97A-G128A-Y217Q-T55P G211R-N212S-K213V 59 G97A-G128A-Y217Q-N61P-S63HL75S-N76Y 60 G97A-G128A-Y217Q-N61P-S63H P86S-S87G-A88V 61G97A-G128A-Y217Q-N61P-S63H S87G-A88V-S89A 62 G97A-G128A-Y217Q-N61P-S63HS87T-A88L-S89G 63 G97A-G128A-Y217Q-N61P-S63H P129Q-S130G-G131S 64G97A-G128A-Y217Q-N61P-S63H V203Y 65 G97A-G128A-Y217Q-N61P-S63HG211R-N212S-K213V 66 G97A-G128A-Y217Q-Q59S-N61P L75S-N76Y 67G97A-G128A-Y217Q-Q59S-N61P P86S-S87G-A88V 68 G97A-G128A-Y217Q-Q59S-N61PS87G-A88V-S89A 69 G97A-G128A-Y217Q-Q59S-N61P S87T-A88L-S89G 70G97A-G128A-Y217Q-Q59S-N61P P129Q-S130G-G131S 71G97A-G128A-Y217Q-Q59S-N61P V203Y 72 G97A-G128A-Y217Q-Q59S-N61PG211R-N212S-K213V 73 G97A-G128A-Y217Q-L75S-N76Y P86S-S87G-A88V 74G97A-G128A-Y217Q-L75S-N76Y S87G-A88V-S89A 75 G97A-G128A-Y217Q-L75S-N76YS87T-A88L-S89G 76 G97A-G128A-Y217Q-L75S-N76Y P129Q-S130G-G131S 77G97A-G128A-Y217Q-L75S-N76Y V203Y 78 G97A-G128A-Y217Q-L75S-N76YG211R-N212S-K213V 79 G97A-G128A-Y217Q-P86S-S87G-A88V P129Q-S130G-G131S80 G97A-G128A-Y217Q-P86S-S87G-A88V V203Y 81G97A-G128A-Y217Q-P86S-S87G-A88V G211R-N212S-K213V 82G97A-G128A-Y217Q-S87G-A88V-S89A P129Q-S130G-G131S 83G97A-G128A-Y217Q-S87G-A88V-S89A V203Y 84 G97A-G128A-Y217Q-S87G-A88V-S89AG211R-N212S-K213V 85 G97A-G128A-Y217Q-S87T-A88L-S89G P129Q-S130G-G131S86 G97A-G128A-Y217Q-S87T-A88L-S89G V203Y 87G97A-G128A-Y217Q-S87T-A88L-S89G G211R-N212S-K213V 88G97A-G128A-Y217Q-P129Q-S130G-G131S V203Y 89G97A-G128A-Y217Q-P129Q-S130G-G131S G211R-N212S-K213V 90G97A-G128A-Y217Q-V203Y G211R-N212S-K213V

TABLE 11-8 Primers Used to Create RCL5 Combinatorial Variants Common 3′& 5′ Gene Mutations Gene Flanking Primer SEQ Introduced Fragments PrimerNames Name ID NO: T22N-S24A 3′ P4976 P5432 535 5′ P4974 P5433 536S24G-N25G 3′ P4976 P5434 537 5′ P4974 P5435 538 S24R 3′ P4976 P5436 5395′ P4974 P5437 540 G23A-S24G-N25G 3′ P4976 P5438 541 5′ P4974 P5439 542N61P-N62S 3′ P4976 P5440 543 5′ P4974 P5441 544 T55P 3′ P4976 P5442 5455′ P4974 P5443 546 N61P-S63H 3′ P4976 P5444 547 5′ P4974 P5445 548Q59S-N61P 3′ P4976 P5446 549 5′ P4974 P5447 550 L75S-N76Y 3′ P4976 P5448551 5′ P4974 P5449 552 P86S-S87G-A88V 3′ P4976 P5450 553 5′ P4974 P5451554 S87G-A88V-S89A 3′ P4976 P5452 555 5′ P4974 P5453 556 S87T-A88L-S89G3′ P4976 P5454 557 5′ P4974 P5455 558 P129Q-S130G-G131S 3′ P4976 P5456559 5′ P4974 P5457 560 V203Y 3′ P4976 P5458 561 5′ P4974 P5459 562G211R-N212S-K213V 3′ P4976 P5460 563 5′ P4974 P5461 564

To create each mutant, two PCR reactions were carried out using eitherthe common 3′ gene-flanking primer (P4976, CCTCTCGGTTATGAGTTAGTTC; SEQID NO:61) and the mutagenic primer, or the common 5′gene-flanking primer(P4974, GCCTCACATTTGTGCCACCTA; SEQ ID NO:60) and mutagenic primer asshown for each library in Table 11-8. These PCR reactions generated twoPCR fragments, one encoding the 5′ half of the mutant BPN′ gene (5′ genefragment) and the other encoding the 3′ half of the mutant BPN′ gene (3′gene fragment). Each PCR amplification reaction contained 30 pmol ofeach primer and 100 ng of the parent molecules listed in Table 11-7.Amplifications were carried out using Vent DNA polymerase (NEB). The PCRreaction (20 μL) was initially heated at 95° C. for 2.5 min followed by30 cycles of denaturation at 94° C. for 15 sec., annealing at 55° C. for15 sec. and extension at 72° C. for 40 sec. Following amplification, the5′ and 3′ gene fragments were gel-purified by the QIAGEN® gel-bandpurification kit, mixed (50 ng of each fragment) and amplified by PCRonce again using the primers P4973 (SEQ ID NO:62) and P4950 (SEQ IDNO:63) to generate the full-length gene fragment. The PCR conditionswere same as described above, except the extension phase, which wascarried out at 72° C. for 2 min. The full-length DNA fragment wasgel-purified by the QIAGEN® gel-band purification kit, digested by theBamHI and HindIII restriction enzymes and ligated with the pHPLT-BPN′partial opt that also was digested with the same restriction enzymes.Ligation mixtures were amplified using rolling circle amplification byIllustra Templiphi kit according to the manufacturer's instructions (GEHealthcare) to generate multimeric DNA for transformation into Bacillussubtilis. For this purpose, 1 μl of the ligation mixture was mixed with5 μl of the sample buffer, heated to 95° C. for 3 min and cooled on ice.Next, 5 μl of the reaction buffer and 0.2 μl of the enzyme were added toeach tube, followed by incubation at 30° C. for 10 hours. Products ofthe rolling circle amplification were diluted 100 times and used totransform B. subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo).An aliquot of the transformation mix was plated on LB plates containing1.6% skim milk and 10 μg/mL neomycin and incubated overnight at 37° C.Subsequently, the colonies with halos were inoculated in 120 μl of LBmedia containing 10 μg/mL neomycin.

RCL 6 Combinatorial Libraries

“RCL6” refers to a group of combinatorial libraries created by PCRfusion using several BPN′ mutants as parent (template) molecules. Amixture of BPN′ mutants were used as templates (parent molecules) in theconstruction of each of these libraries. The five different mixes ofparent molecules used to create these libraries are provided in Table11-9, and the mutations introduced in each library are listed in Table11-10.

To create each mutant, two PCR reactions were carried out using eitherthe common 3′ gene-flanking primer (P4976, SEQ ID NO:61) and themutagenic primer, or the common 5′gene-flanking primer (P4974, SEQ IDNO:60) and mutagenic primer as shown for each library in Table 11-10.These PCR reactions generated two PCR fragments, one encoding the 5′half of the mutant BPN′ gene (5′ gene fragment) and the other encodingthe 3′ half of the mutant BPN′ gene (3′ gene fragment). Each PCRamplification reaction contained 30 pmol of each primer and 100 ng ofthe parent molecules listed in Table 11-9 Amplifications were carriedout using Vent DNA polymerase (NEB). The PCR reaction (20 μL) wasinitially heated at 95° C. for 2.5 min followed by 30 cycles ofdenaturation at 94° C. for 15 sec., annealing at 55° C. for 15 sec. andextension at 72° C. for 40 sec. Following amplification, the 5′ and 3′gene fragments were gel-purified by the QIAGEN® gel-band purificationkit, mixed (50 ng of each fragment) and amplified by PCR once againusing the primers P4973 (SEQ ID NO:62) and P4950 (SEQ ID NO:63) togenerate the full-length gene fragment. The PCR conditions were same asdescribed above, except the extension phase, which was carried out at72° C. for 2 min. The full-length DNA fragment was gel-purified by theQIAGEN® gel-band purification kit, digested using BamHI and HindIIIrestriction enzymes and ligated with the pHPLT-BPN′ partial opt vectorthat also was digested with the same restriction enzymes. Ligationmixtures were amplified using rolling circle amplification by IllustraTempliphi kit according to the manufacturer's instructions (GEHealthcare) to generate multimeric DNA for transformation into Bacillussubtilis. For this purpose, 1 μl of the ligation mixture was mixed with5 μl of the sample buffer, heated to 95° C. for 3 min and cooled on ice.Next, 5 μl of the reaction buffer and 0.2 μl of the enzyme were added toeach tube, followed by incubation at 30° C. for 10 hours. Products ofthe rolling circle amplification were diluted 100 times and used totransform B. subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo).An aliquot of the transformation mix was plated on LB plates containing1.6% skim milk and 10 μg/mL neomycin and incubated overnight at 37° C.Subsequently, the colonies with halos were inoculated in 120 μl of LBmedia containing 10 μg/mL neomycin.

TABLE 11-9 Parent Molecules of BPN′ Used to Create RCL6 Libraries Mixesof Parent Molecules Mix 1 Mix 2 Mix 3 Mix 4 Mix 5 G97A-G128A-G97A-G128A- G97A-G128A- G97A-G128A- G97A-G128A- Y217Q-S24G- Y217Q-T55PY217Q-L75S- Y217Q-P86S- Y217Q-P129Q- N25G N76Y S87G-A88V S130G-G131SG97A-G128A- G97A-G128A- G97A-G128A- Y217Q-S24R Y217Q-N61P- Y217Q-S87G-S63H A88V-S89A G97A-G128A- G97A-G128A- G97A-G128A- Y217Q-G23A-Y217Q-Q59S- Y217Q-S87T- S24G-N25G N61P A88L-S89G

TABLE 11-10 Mutations Introduced in the RCL6 Libraries Common 5′ & 3′Gene Flanking Mutagenic SEQ Gene Primer Primer ID Mutations IntroducedFragment Names Name NO: V68C, A69G 3′ P4976 P5462 565 5′ P4974 P5463 566V72I, A73G, delA74, L75S 3′ P4976 P5464 567 5′ P4974 P5465 568 L75H,N76G 3′ P4976 P5466 569 5′ P4974 P5467 570 L75R, N76G, N77S 3′ P4976P5468 571 5′ P4974 P5469 572 L75G, N76G, N77G 3′ P4976 P5470 573 5′P4974 P5471 574 A92G 3′ P4976 P5472 575 5′ P4974 P5473 576 delV93, K94S,V95C, 3′ P4976 P5474 577 L96S 5′ P4974 P5475 578 V121I_I122S_N123C 3′P4976 P5476 579 5′ P4974 P5477 580 V121L_N123C 3′ P4976 P5478 581 5′P4974 P5479 582 I122C_N123S_M124L 3′ P4976 P5480 583 5′ P4974 P5481 584N123C 3′ P4976 P5482 585 5′ P4974 P5483 586 M124I 3′ P4976 P5484 587 5′P4974 P5485 588 M124V 3′ P4976 P5486 589 5′ P4974 P5487 590 M124V-L126A3′ P4976 P5488 591 5′ P4974 P5489 592 L126F, delP129 3′ P4976 P5490 5935′ P4974 P5491 594 G127Y 3′ P4976 P5492 595 5′ P4974 P5493 596G127S_P129D 3′ P4976 P5494 597 5′ P4974 P5495 598 G127N, P129R 3′ P4976P5496 599 5′ P4974 P5497 600 G128N, insS, P129S 3′ P4976 P5498 601 5′P4974 P5499 602 G128S_P129V 3′ P4976 P5500 603 5′ P4974 P5501 604 G128S,P129D 3′ P4976 P5502 605 5′ P4974 P5503 606 G128S, P129G 3′ P4976 P5504607 5′ P4974 P5505 608 H128H, P129Y 3′ P4976 P5506 609 5′ P4974 P5507610 P129D 3′ P4976 P5508 611 5′ P4974 P5509 612 P129E 3′ P4976 P5510 6135′ P4974 P5511 614 P129V 3′ P4976 P5512 615 5′ P4974 P5513 616 P129G,delS130 3′ P4976 P5514 617 5′ P4974 P5515 618 P129H, delS130, S132N 3′P4976 P5516 619 5′ P4974 P5517 620 A134T 3′ P4976 P5518 621 5′ P4974P5519 622 G97R, insG, A98C 3′ P4976 P5520 623 5′ P4974 P5521 624 A98G,D99G 3′ P4976 P5522 625 5′ P4974 P5523 626 A98G, insR 3′ P4976 P5524 6275′ P4974 P5525 628 A98D, D99G 3′ P4976 P5526 629 5′ P4974 P5527 630A98H, D99G, G100D 3′ P4976 P5528 631 5′ P4974 P5529 632 D99R, insN 3′P4976 P5530 633 5′ P4974 P5531 634 D99V, S101D 3′ P4976 P5532 635 5′P4974 P5533 636 D99C, insS 3′ P4976 P5534 637 5′ P4974 P5535 638 G100S3′ P4976 P5536 639 5′ P4974 P5537 640 G100S, S101V 3′ P4976 P5538 641 5′P4974 P5539 642 G100D 3′ P4976 P5540 643 5′ P4974 P5541 644 G100N 3′P4976 P5542 645 5′ P4974 P5543 646 S100N, S101L 3′ P4976 P5544 647 5′P4974 P5545 648 S101G 3′ P4976 P5546 649 5′ P4974 P5547 650 S101D 3′P4976 P5548 651 5′ P4974 P5549 652 S101V, Q103N 3′ P4976 P5550 653 5′P4974 P5551 654 S101E 3′ P4976 P5552 655 5′ P4974 P5553 656 A116S,N117G, N118R 3′ P4976 P5554 657 5′ P4974 P5555 658 A116G, N117R 3′ P4976P5556 659 5′ P4974 P5557 660 A116N, N117S, N118G 3′ P4976 P5558 661 5′P4974 P5559 662 M222Q 3′ P4976 P5560 663 5′ P4974 P5561 664 S24R 3′P4976 P5562 665 5′ P4974 P5563 666 N25Y 3′ P4976 P5564 667 5′ P4974P5565 668 P52D 3′ P4976 P5566 669 5′ P4974 P5567 670 S63T 3′ P4976 P5568671 5′ P4974 P5569 672 N61E 3′ P4976 P5570 673 5′ P4974 P5571 674 N61P3′ P4976 P5572 675 5′ P4974 P5573 676 N62Q 3′ P4976 P5574 677 5′ P4974P5575 678 N62D 3′ P4976 P5576 679 5′ P4974 P5577 680 S63Q 3′ P4976 P5578681 5′ P4974 P5579 682 V68A 3′ P4976 P5580 683 5′ P4974 P5581 684 S87D3′ P4976 P5582 685 5′ P4974 P5583 686 L96T 3′ P4976 P5584 687 5′ P4974P5585 688 L126A 3′ P4976 P5586 689 5′ P4974 P5587 690 L126T 3′ P4976P5588 691 5′ P4974 P5589 692 S125A 3′ P4976 P5590 693 5′ P4974 P5591 694S130P 3′ P4976 P5592 695 5′ P4974 P5593 696 P129L 3′ P4976 P5594 697 5′P4974 P5595 698 P129E 3′ P4976 P5596 699 5′ P4974 P5597 700 P129S 3′P4976 P5598 701 5′ P4974 P5599 702 P40E 3′ P4976 P5600 703 5′ P4974P5601 704 Y6Q 3′ P4976 P5602 705 5′ P4974 P5603 706 G102A 3′ P4976 P5604707 5′ P4974 P5605 708 S101N 3′ P4976 P5606 709 5′ P4974 P5607 710 G100E3′ P4976 P5608 711 5′ P4974 P5609 712 I115V 3′ P4976 P5610 713 5′ P4974P5611 714 A144K 3′ P4976 P5612 715 5′ P4974 P5613 716 S145D 3′ P4976P5614 717 5′ P4974 P5615 718 S159K 3′ P4976 P5616 719 5′ P4974 P5617 720S162K 3′ P4976 P5618 721 5′ P4974 P5619 722 V147P 3′ P4976 P5620 723 5′P4974 P5621 724 S161P 3′ P4976 P5622 725 5′ P4974 P5623 726 A187D 3′P4976 P5624 727 5′ P4974 P5625 728 F189D 3′ P4976 P5626 729 5′ P4974P5627 730 L267V 3′ P4976 P5628 731 5′ P4974 P5629 732 Q206E 3′ P4976P5630 733 5′ P4974 P5631 734 K213T 3′ P4976 P5632 735 5′ P4974 P5633 736K213L 3′ P4976 P5634 737 5′ P4974 P5635 738 K265N 3′ P4976 P5636 739 5′P4974 P5637 740 N240K 3′ P4976 P5638 741 5′ P4974 P5639 742 P239R 3′P4976 P5640 743 5′ P4974 P5641 744 T242R 3′ P4976 P5642 745 5′ P4974P5643 746 S89Y 3′ P4976 P5644 747 5′ P4974 P5645 748 P129Q 3′ P4976P5646 749 5′ P4974 P5647 750 G211T 3′ P4976 P5648 751 5′ P4974 P5649 752I111V 3′ P4976 P5650 753 5′ P4974 P5651 754RCL 7 Combinatorial Variants

“RCL7” refers to a set of combinatorial variants created by PCR fusionusing several BPN′ mutants as parent (template) plasmid. The mutationsintroduced in each parent plasmid are listed in Table 11-11, and themutagenic primers used to create the mutants are described in Table11-10.

To create each mutant, two PCR reactions were carried out using eitherthe common 3′ gene-flanking primer (P4976, SEQ ID NO:61) and themutagenic primer, or the common 5′gene-flanking primer (P4974, SEQ IDNO:60) and mutagenic primer as shown for each library in Table 11-10.These PCR reactions generated two PCR fragments, one encoding the 5′half of the mutant BPN′ gene (5′ gene fragment) and the other encodingthe 3′ half of the mutant BPN′ gene (3′ gene fragment). Each PCRamplification reaction contained 30 pmol of each primer and 100 ng ofthe parent molecules listed in Table 11-11. Amplifications were carriedout using Vent DNA polymerase (NEB). The PCR reaction (20 μL) wasinitially heated at 95° C. for 2.5 min followed by 30 cycles ofdenaturation at 94° C. for 15 sec., annealing at 55° C. for 15 sec. andextension at 72° C. for 40 sec. Following amplification, the 5′ and 3′gene fragments were gel-purified using a QIAGEN® gel-band purificationkit, mixed (50 ng of each fragment) and amplified by PCR once againusing the primers P4973 (SEQ ID NO:62) and P4950 (SEQ ID NO:63) togenerate the full-length gene fragment. The PCR conditions were same asdescribed above, except the extension phase, which was carried out at72° C. for 2 min. The full-length DNA fragment was gel-purified using aQIAGEN® gel-band purification kit, digested using BamHI and HindIIIrestriction enzymes, and ligated with the pHPLT-BPN′ partial opt thatalso was digested with the same restriction enzymes. Ligation mixtureswere amplified using rolling circle amplification by Illustra Templiphikit according to the manufacturer's instructions (GE Healthcare) togenerate multimeric DNA for transformation into Bacillus subtilis. Forthis purpose, 1 μl of the ligation mixture was mixed with 5 μl of thesample buffer, heated to 95° C. for 3 min and cooled on ice. Next, 5 μlof the reaction buffer and 0.2 μl of the enzyme were added to each tube,followed by incubation at 30° C. for 10 hours. Products of the rollingcircle amplification were diluted 100 times and used to transform B.subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). An aliquot ofthe transformation mix was plated on LB plates containing 1.6% skim milkand 10 μg/mL neomycin and incubated overnight at 37° C. Subsequently,the colonies with halos were inoculated in 120 μl of LB media containing10 μg/mL neomycin.

TABLE 11-11 List of Parent Plasmids and Introduced Mutations in the RCL7variants Combi- natorial Mutation(s) Variant # Parent Plasmid Introduced1 G97A-G128A-Y217Q-T55P S24R 2 G97A-G128A-Y217Q-N61E S24R 3G97A-G128A-Y217Q-N61P-S63H S24R 4 G97A-G128A-Y217Q-L75S-N76Y S24R 5G97A-G128A-Y217Q-S87T-A88L- S24R S89G 6 G97A-G128A-Y217Q-S89Y S24R 7G97A-G128A-Y217Q-I111V S24R 8 G97A-G128A-Y217Q-I115V S24R 9G97A-G128A-Y217Q-P129Q-S130G- S24R G131S 10 G97A-G128A-Y217Q-P129Q S24R11 G97A-G128A-Y217Q-A134T S24R 12 G97A-G128A-Y217Q-A144K S24R 13G97A-G128A-Y217Q-S145D S24R 14 G97A-G128A-Y217Q-S159K S24R 15G97A-G128A-Y217Q-S162K S24R 16 G97A-G128A-Y217Q-S161P-S78N S24R 17G97A-G128A-Y217Q-V203Y S24R 18 G97A-G128A-Y217Q-G211T-S78N S24R 19G97A-G128A-Y217Q-K213T S24R 20 G97A-G128A-Y217Q-P239R S24R 21G97A-G128A-Y217Q-N240K S24R 22 G97A-G128A-Y217Q-L267V-S78N S24R 23G97A-G128A-Y217Q-A273S-S78N S24R 24 G97A-G128A-Y217Q-L75S-N76Y T55P 25G97A-G128A-Y217Q-S87T-A88L- T55P S89G 26 G97A-G128A-Y217Q-S89Y T55P 27G97A-G128A-Y217Q-I111V T55P 28 G97A-G128A-Y217Q-I115V T55P 29G97A-G128A-Y217Q-P129Q-S130G- T55P G131S 30 G97A-G128A-Y217Q-P129Q T55P31 G97A-G128A-Y217Q-A134T T55P 32 G97A-G128A-Y217Q-A144K T55P 33G97A-G128A-Y217Q-S145D T55P 34 G97A-G128A-Y217Q-S159K T55P 35G97A-G128A-Y217Q-S162K T55P 36 G97A-G128A-Y217Q-S161P T55P 37G97A-G128A-Y217Q-V203Y T55P 38 G97A-G128A-Y217Q-G211T T55P 39G97A-G128A-Y217Q-K213T T55P 40 G97A-G128A-Y217Q-P239R T55P 41G97A-G128A-Y217Q-N240K T55P 42 G97A-G128A-Y217Q-L267V T55P 43G97A-G128A-Y217Q-A273S T55P 44 G97A-G128A-Y217Q-L75S-N76Y N61E 45G97A-G128A-Y217Q-S87T-A88L- N61E S89G 46 G97A-G128A-Y217Q-S89Y N61E 47G97A-G128A-Y217Q-I111V N61E 48 G97A-G128A-Y217Q-I115V N61E 49G97A-G128A-Y217Q-P129Q-S130G- N61E G131S 50 G97A-G128A-Y217Q-P129Q N61E51 G97A-G128A-Y217Q-A134T N61E 52 G97A-G128A-Y217Q-A144K N61E 53G97A-G128A-Y217Q-S145D N61E 54 G97A-G128A-Y217Q-S159K N61E 55G97A-G128A-Y217Q-S162K N61E 56 G97A-G128A-Y217Q-S161P N61E 57G97A-G128A-Y217Q-V203Y N61E 58 G97A-G128A-Y217Q-G211T N61E 59G97A-G128A-Y217Q-K213T N61E 60 G97A-G128A-Y217Q-P239R N61E 61G97A-G128A-Y217Q-N240K N61E 62 G97A-G128A-Y217Q-L267V N61E 63G97A-G128A-Y217Q-A273S N61E 64 G97A-G128A-Y217Q-L75S-N76Y N61P-S63H 65G97A-G128A-Y217Q-S87T-A88L- N61P-S63H S89G 66 G97A-G128A-Y217Q-S89YN61P-S63H 67 G97A-G128A-Y217Q-I111V N61P-S63H 68 G97A-G128A-Y217Q-I115VN61P-S63H 69 G97A-G128A-Y217Q-P129Q-S130G- N61P-S63H G131S 70G97A-G128A-Y217Q-P129Q N61P-S63H 71 G97A-G128A-Y217Q-A134T N61P-S63H 72G97A-G128A-Y217Q-A144K N61P-S63H 73 G97A-G128A-Y217Q-S145D N61P-S63H 74G97A-G128A-Y217Q-S159K N61P-S63H 75 G97A-G128A-Y217Q-S162K N61P-S63H 76G97A-G128A-Y217Q-S161P N61P-S63H 77 G97A-G128A-Y217Q-V203Y N61P-S63H 78G97A-G128A-Y217Q-G211T N61P-S63H 79 G97A-G128A-Y217Q-K213T N61P-S63H 80G97A-G128A-Y217Q-P239R N61P-S63H 81 G97A-G128A-Y217Q-N240K N61P-S63H 82G97A-G128A-Y217Q-L267V N61P-S63H 83 G97A-G128A-Y217Q-A273S N61P-S63H 84G97A-G128A-Y217Q-S87T-A88L- L75S-N76Y S89G 85 G97A-G128A-Y217Q-S89YL75S-N76Y 86 G97A-G128A-Y217Q-I111V L75S-N76Y 87 G97A-G128A-Y217Q-I115VL75S-N76Y 88 G97A-G128A-Y217Q-P129Q-S130G- L75S-N76Y G131S 89G97A-G128A-Y217Q-P129Q L75S-N76Y 90 G97A-G128A-Y217Q-A134T L75S-N76Y 91G97A-G128A-Y217Q-A144K L75S-N76Y 92 G97A-G128A-Y217Q-S145D L75S-N76Y 93G97A-G128A-Y217Q-S159K L75S-N76Y 94 G97A-G128A-Y217Q-S162K L75S-N76Y 95G97A-G128A-Y217Q-S161P L75S-N76Y 96 G97A-G128A-Y217Q-V203Y L75S-N76Y 97G97A-G128A-Y217Q-G211T L75S-N76Y 98 G97A-G128A-Y217Q-K213T L75S-N76Y 99G97A-G128A-Y217Q-P239R L75S-N76Y 100 G97A-G128A-Y217Q-N240K L75S-N76Y101 G97A-G128A-Y217Q-L267V L75S-N76Y 102 G97A-G128A-Y217Q-A273SL75S-N76Y 103 G97A-G128A-Y217Q-I111V S87T-A88L-S89G 104G97A-G128A-Y217Q-I115V S87T-A88L-S89G 105 G97A-G128A-Y217Q-P129Q-S130G-S87T-A88L-S89G G131S 106 G97A-G128A-Y217Q-P129Q S87T-A88L-S89G 107G97A-G128A-Y217Q-A134T S87T-A88L-S89G 108 G97A-G128A-Y217Q-A144KS87T-A88L-S89G 109 G97A-G128A-Y217Q-S145D S87T-A88L-S89G 110G97A-G128A-Y217Q-S159K S87T-A88L-S89G 111 G97A-G128A-Y217Q-S162KS87T-A88L-S89G 112 G97A-G128A-Y217Q-S161P S87T-A88L-S89G 113G97A-G128A-Y217Q-V203Y S87T-A88L-S89G 114 G97A-G128A-Y217Q-G211TS87T-A88L-S89G 115 G97A-G128A-Y217Q-K213T S87T-A88L-S89G 116G97A-G128A-Y217Q-P239R S87T-A88L-S89G 117 G97A-G128A-Y217Q-N240KS87T-A88L-S89G 118 G97A-G128A-Y217Q-L267V S87T-A88L-S89G 119G97A-G128A-Y217Q-A273S S87T-A88L-S89G 120 G97A-G128A-Y217Q-I111V S89Y121 G97A-G128A-Y217Q-I115V S89Y 122 G97A-G128A-Y217Q-P129Q-S130G- S89YG131S 123 G97A-G128A-Y217Q-P129Q S89Y 124 G97A-G128A-Y217Q-A134T S89Y125 G97A-G128A-Y217Q-A144K S89Y 126 G97A-G128A-Y217Q-S145D S89Y 127G97A-G128A-Y217Q-S159K S89Y 128 G97A-G128A-Y217Q-S162K S89Y 129G97A-G128A-Y217Q-S161P S89Y 130 G97A-G128A-Y217Q-V203Y S89Y 131G97A-G128A-Y217Q-G211T S89Y 132 G97A-G128A-Y217Q-K213T S89Y 133G97A-G128A-Y217Q-P239R S89Y 134 G97A-G128A-Y217Q-N240K S89Y 135G97A-G128A-Y217Q-L267V S89Y 136 G97A-G128A-Y217Q-A273S S89Y 137G97A-G128A-Y217Q-P129Q-S130G- I111V G131S 138 G97A-G128A-Y217Q-P129QI111V 139 G97A-G128A-Y217Q-A134T I111V 140 G97A-G128A-Y217Q-A144K I111V141 G97A-G128A-Y217Q-S145D I111V 142 G97A-G128A-Y217Q-S159K I111V 143G97A-G128A-Y217Q-S162K I111V 144 G97A-G128A-Y217Q-S161P I111V 145G97A-G128A-Y217Q-V203Y I111V 146 G97A-G128A-Y217Q-G211T I111V 147G97A-G128A-Y217Q-K213T I111V 148 G97A-G128A-Y217Q-P239R I111V 149G97A-G128A-Y217Q-N240K I111V 150 G97A-G128A-Y217Q-L267V I111V 151G97A-G128A-Y217Q-A273S I111V 152 G97A-G128A-Y217Q-P129Q-S130G- I115VG131S 153 G97A-G128A-Y217Q-P129Q I115V 154 G97A-G128A-Y217Q-A134T I115V155 G97A-G128A-Y217Q-A144K I115V 156 G97A-G128A-Y217Q-S145D I115V 157G97A-G128A-Y217Q-S159K I115V 158 G97A-G128A-Y217Q-S162K I115V 159G97A-G128A-Y217Q-S161P I115V 160 G97A-G128A-Y217Q-V203Y I115V 161G97A-G128A-Y217Q-G211T I115V 162 G97A-G128A-Y217Q-K213T I115V 163G97A-G128A-Y217Q-P239R I115V 164 G97A-G128A-Y217Q-N240K I115V 165G97A-G128A-Y217Q-L267V I115V 166 G97A-G128A-Y217Q-A273S I115V 167G97A-G128A-Y217Q-A144K P129Q-S130G- G131S 168 G97A-G128A-Y217Q-S145DP129Q-S130G- G131S 169 G97A-G128A-Y217Q-S159K P129Q-S130G- G131S 170G97A-G128A-Y217Q-S162K P129Q-S130G- G131S 171 G97A-G128A-Y217Q-S161PP129Q-S130G- G131S 172 G97A-G128A-Y217Q-V203Y P129Q-S130G- G131S 173G97A-G128A-Y217Q-G211T P129Q-S130G- G131S 174 G97A-G128A-Y217Q-K213TP129Q-S130G- G131S 175 G97A-G128A-Y217Q-P239R P129Q-S130G- G131S 176G97A-G128A-Y217Q-N240K P129Q-S130G- G131S 177 G97A-G128A-Y217Q-L267VP129Q-S130G- G131S 178 G97A-G128A-Y217Q-A273S P129Q-S130G- G131S 179G97A-G128A-Y217Q-A144K P129Q 180 G97A-G128A-Y217Q-S145D P129Q 181G97A-G128A-Y217Q-S159K P129Q 182 G97A-G128A-Y217Q-S162K P129Q 183G97A-G128A-Y217Q-S161P P129Q 184 G97A-G128A-Y217Q-V203Y P129Q 185G97A-G128A-Y217Q-G211T P129Q 186 G97A-G128A-Y217Q-K213T P129Q 187G97A-G128A-Y217Q-P239R P129Q 188 G97A-G128A-Y217Q-N240K P129Q 189G97A-G128A-Y217Q-L267V P129Q 190 G97A-G128A-Y217Q-A273S P129Q 191G97A-G128A-Y217Q-A144K A134T 192 G97A-G128A-Y217Q-S145D A134T 193G97A-G128A-Y217Q-S159K A134T 194 G97A-G128A-Y217Q-S162K A134T 195G97A-G128A-Y217Q-S161P A134T 196 G97A-G128A-Y217Q-V203Y A134T 197G97A-G128A-Y217Q-G211T A134T 198 G97A-G128A-Y217Q-K213T A134T 199G97A-G128A-Y217Q-P239R A134T 200 G97A-G128A-Y217Q-N240K A134T 201G97A-G128A-Y217Q-L267V A134T 202 G97A-G128A-Y217Q-A273S A134T 203G97A-G128A-Y217Q-S159K A144K 204 G97A-G128A-Y217Q-S162K A144K 205G97A-G128A-Y217Q-S161P A144K 206 G97A-G128A-Y217Q-V203Y A144K 207G97A-G128A-Y217Q-G211T A144K 208 G97A-G128A-Y217Q-K213T A144K 209G97A-G128A-Y217Q-P239R A144K 210 G97A-G128A-Y217Q-N240K A144K 211G97A-G128A-Y217Q-L267V A144K 212 G97A-G128A-Y217Q-A273S A144K 213G97A-G128A-Y217Q-S159K S145D 214 G97A-G128A-Y217Q-S162K S145D 215G97A-G128A-Y217Q-S161P S145D 216 G97A-G128A-Y217Q-V203Y S145D 217G97A-G128A-Y217Q-G211T S145D 218 G97A-G128A-Y217Q-K213T S145D 219G97A-G128A-Y217Q-P239R S145D 220 G97A-G128A-Y217Q-N240K S145D 221G97A-G128A-Y217Q-L267V S145D 222 G97A-G128A-Y217Q-A273S S145D 223G97A-G128A-Y217Q-V203Y S159K 224 G97A-G128A-Y217Q-G211T S159K 225G97A-G128A-Y217Q-K213T S159K 226 G97A-G128A-Y217Q-P239R S159K 227G97A-G128A-Y217Q-N240K S159K 228 G97A-G128A-Y217Q-L267V S159K 229G97A-G128A-Y217Q-A273S S159K 230 G97A-G128A-Y217Q-V203Y S162K 231G97A-G128A-Y217Q-G211T S162K 232 G97A-G128A-Y217Q-K213T S162K 233G97A-G128A-Y217Q-P239R S162K 234 G97A-G128A-Y217Q-N240K S162K 235G97A-G128A-Y217Q-L267V S162K 236 G97A-G128A-Y217Q-A273S S162K 237G97A-G128A-Y217Q-V203Y S161P 238 G97A-G128A-Y217Q-G211T S161P 239G97A-G128A-Y217Q-K213T S161P 240 G97A-G128A-Y217Q-239R S161P 241G97A-G128A-Y217Q-N240K S161P 242 G97A-G128A-Y217Q-L267V S161P 243G97A-G128A-Y217Q-A273S S161P 244 G97A-G128A-Y217Q-G211T V203Y 245G97A-G128A-Y217Q-K213T V203Y 246 G97A-G128A-Y217Q-P239R V203Y 247G97A-G128A-Y217Q-N240K V203Y 248 G97A-G128A-Y217Q-L267V V203Y 249G97A-G128A-Y217Q-A273S V203Y 250 G97A-G128A-Y217Q-P239R G211T 251G97A-G128A-Y217Q-N240K G211T 252 G97A-G128A-Y217Q-L267V G211T 253G97A-G128A-Y217Q-A273S G211T 254 G97A-G128A-Y217Q-P239R K213T 255G97A-G128A-Y217Q-N240K K213T 256 G97A-G128A-Y217Q-L267V K213T 257G97A-G128A-Y217Q-A273S K213T 258 G97A-G128A-Y217Q-L267V P239R 259G97A-G128A-Y217Q-A273S P239R 260 G97A-G128A-Y217Q-L267V N240K 261G97A-G128A-Y217Q-A273S N240K

Example 12

Table of Detergents

The compositions of the detergents used in the assays for Part I Example12 are shown in Table 12-1. BPN′ variant protein samples were added tothe detergent compositions as described in Part I Example 1 to assay forthe various properties listed.

TABLE 12-1 Composition of Detergents Used in the Assays to Test BPN′Variants Composition (wt % of Composition) Ingredient 1 2 3 C₁₂₋₁₅Alkylethoxy(1.8)sulphate 14.7 11.6 C_(11.8) Alkylbenzene sulfonate 4.311.6 8.3 C₁₆₋₁₇ Branched alkyl sulphate 1.7 1.29 C₁₂₋₁₄Alkyl-9-ethoxylate 0.9 1.07 C₁₂ dimethylamine oxide 0.6 0.64 Citric acid3.5 0.65 3 C₁₂₋₁₈ fatty acid 1.5 2.32 3.6 Sodium Borate (Borax) 2.5 2.461.2 Sodium C₁₂₋₁₄ alkyl ethoxy 3 sulfate 2.9 C₁₄₋₁₅ alkyl 7-ethoxylate4.2 C₁₂₋₁₄ Alkyl-7-ethoxylate 1.7 Ca formate 0.09 0.09 A compound havingthe following general structure: 1.2bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)- bis((C₂H₅O)(C₂H₄O)n),wherein n = from 20 to 30, and x = from 3 to 8, or sulphated orsulphonated variants thereof Random graft co-polymer¹ 1.46 0.5Ethoxylated Polyethylenimine ² 1.5 1.29 Diethylene triamine pentaaceticacid 0.34 0.64 Diethylene triamine penta(methylene phosphonic acid) 0.3Tinopal AMS-GX 0.06 Tinopal CBS-X 0.2 0.17 Amphiphilic alkoxylatedgrease cleaning polymer ³ 1.28 1 0.4 Ethanol 2 1.58 1.6 Propylene Glycol3.9 3.59 1.3 Diethylene glycol 1.05 1.54 Polyethylene glycol 0.06 0.04Monoethanolamine 3.05 2.41 0.4 NaOH 2.44 1.8 Sodium Cumene Sulphonate 1Sodium Formate 0.11 Water, Aesthetics (Dyes, perfumes) and Minorsbalance balance balance (Enzymes, solvents, structurants) ¹“Random graftcopolymer” is a polyvinyl acetate grafted polyethylene oxide copolymerhaving a polyethylene oxide backbone and multiple polyvinyl acetate sidechains. The molecular weight of the polyethylene oxide backbone is about6000 and the weight ratio of the polyethylene oxide to polyvinyl acetateis about 40 to 60 and no more than 1 grafting point per 50 ethyleneoxide units. ² Polyethylenimine (MW = 600) with 20 ethoxylate groups per—NH. ³ Amphiphilic alkoxylated grease cleaning polymer is apolyethylenimine (MW = 600) with 24 ethoxylate groups per —NH and 16propoxylate groups per —NH.

Stain Removal Performance of BPN′ Combinatorial Variants

Experiments to evaluate the stain removal performance of BPN′combinatorial variants generated as described in Example 11 wereperformed using BMI stained microswatches. The assay was performed asdescribed in Example 1 (BMI microswatch assay). Table 12-2 providesPerformance Index (PI) values of variants generated from RCL4 libraryusing BMI microswatch assay in Detergent Composition 1 at pH 8 and 16°C. and Detergent Composition 1 at pH 8 and 32° C. and BMI microswatchassay in heat deactivated commercial TIDE® 2× Cold (Proctor & Gamble)detergent at 16° C. and pH 8. Heat inactivation of commercial detergentformulas serves to destroy the endogenous enzymatic activity of anyprotein components while retaining the properties of nonenzymaticcomponents. Heat inactivation of the detergents was performed by placingpre-weighed amounts of liquid detergent (in a glass bottle) in a waterbath at 95° C. for 2 hours. The TIDE® 2× Cold detergent was purchasedfrom local supermarket stores. Both unheated and heated detergents wereassayed within 5 minutes of dissolving the detergent, in order toaccurately determine percentage deactivated. Enzyme activity was testedby AAPF assay. Working solutions were made from the heat inactivatedstock. Appropriate amounts of water hardness and buffer were added tothe detergent solutions to match the desired conditions (Table 12-2).The solutions were mixed by vortexing or inverting the bottles.

TABLE 12-2 Working Detergent Solutions Temp Detergent Hardness Detergent(° C.) g/L pH Buffer Gpg TIDE ® 2X Cold 16, 32 0.98 8 5 mM HEPES 6

The sequences of the variants listed in Table 12-3 are relative toBPN′-v3: G97A-G128A-Y217Q. The PI values are calculated relative toBPN′-v3. All mutants in this list have a PI cutoff equal or greater than0.5 for at least one property tested. “Det. Comp.” means DetergentComposition.

TABLE 12-3 Performance Index Values of Variants Generated from RCL4Library Sequence Sequence Relative to TIDE ® Det. Comp. 1, Det. Comp. 1,Relative to BPN′-v3: G97A- Detergent pH 8, pH 8, 16° pH 8, 32° BPN′G128A-Y217Q 16° C., BMI PI C., BMI PI C., BMI PI S87T-A88L-S89G-S87T-A88L- 1.00 1.12 0.99 G97A-G128A- S89G Y217Q N61P-S63H-G97A-N61P-S63H 1.03 1.12 1.01 G128A-Y217Q S87G-A88V-S89A- S87G-A88V- 1.021.11 0.99 G97A-G128A- S89A Y217Q P86S-S87G-A88V- P86S-S87G- 1.00 1.101.00 G97A-G128A- A88V Y217Q Q59S-N61P-G97A- Q59S-N61P 1.01 1.09 1.00G128A-Y217Q S24G-N25G-G97A- S24G-N25G 0.99 1.09 1.02 G128A-Y217QN61P-N62S-G97A- N61P-N62S 0.99 1.06 0.98 G128A-Y217Q G97A-G128A-P129Q-S130G- 0.96 1.06 0.99 P129Q-S130G- G131S G131S-Y217QL75S-N76Y-G97A- L75S-N76Y 0.99 1.06 1.00 G128A-Y217Q G97A-G128A- V203Y0.99 1.05 1.01 V203Y-Y217Q T55P-G97A-G128A- T55P 0.98 1.04 0.98 Y217QA88V-L90I-G97A- A88V-L90I 0.99 1.04 1.00 G128A-Y217Q G97A-G128A-G211R-N212S- 0.97 1.04 0.98 G211R-N212S- K213V K213V-Y217QG23A-S24G-N25G- G23A-S24G- 0.98 1.04 0.98 G97A-G128A- N25G Y217QT22N-S24A-G97A- T22N-S24A 0.98 1.03 0.97 G128A-Y217Q S24R-G97A-G128A-S24R 0.95 1.02 0.99 Y217Q G97A-A98S-G128A- A98S 0.95 1.02 0.99 Y217QBPN′-v3: G97A- BPN′-v3 1.00 1.00 1.00 G128A-Y217Q G97A-G128A-T158G-S159G 0.95 0.99 0.97 T158G-S159G- Y217Q Q59E-N61P-G97A- Q59E-N61P0.90 0.94 0.90 G128A-Y217Q G97A-A98E-G128A- A98E 0.92 0.91 0.90 Y217Q

The invention includes a protease variant having proteolytic activity,the variant comprising an amino acid sequence having at least 60%, 70%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identityto SEQ ID NO:2 or SEQ ID NO:6 and at least one set of amino acidsubstitutions selected from those in Table 12-3, wherein positions ofthe variant are numbered by correspondence with positions of the SEQ IDNO:2 sequence. Each such protease variant may be an isolated,recombinant, substantially pure, or non-naturally occurring proteasevariant. Also included are compositions, including cleaningcompositions, comprising at least one such protease variant and methodsfor cleaning utilizing at least one such protease variant as describedin greater detail elsewhere herein.

Table 12-4 provides Performance Index (PI) values of variants generatedfrom RCL 5-7 and FS1-3 using BMI microswatch assay in DetergentComposition 1 (from Table 1-3) at 16° C. and pH 8, BMI microswatch assayin Detergent Composition 2 (from Table 1-3) at 16° C. and pH 8, andstability measured in Detergent Composition 3 (from Table 1-3). PIvalues for specific activity by AAPF hydrolysis (Specific AAPF PI) werealso determined. All assays were performed as described in Example 1.The sequences of the variants listed are relative to BPN′-v3:G97A-G128A-Y217Q. PI values were calculated relative to BPN′-v3. Allmutants in this list have a PI cutoff equal or greater than 0.5 for atleast one property tested. PI values less than 0.01 were modified todisplay 0.01 in bold italics.

TABLE 12-4 Performance Index Values of Variants Generated From RCL 5-7and FS1-3 Sequence Source Sequence Relative to Det. Comp. 1, Det. Comp.2, Det. Comp. 3, of Relative to BPN′-v3: G97A- pH 8, 16° C., pH 8, 16°C., Specific Stability Variants BPN′ G128A-Y217Q BMI PI BMI PI AAPF PIPI RCL6 G97A-G128A- P86S-S87G- 1.21 1.14 1.17 0.03 Y217Q-P86S-A88V-A116N- S87G-A88V- N117S-N118G A116N-N117S- N118G FS1 G97A-G128A-S24G-N25G- 1.05 1.12 1.96 1.00 Y217Q-S24G- N61P-S101N N25G-N61P- S101NFS1 G97A-G128A- S24G-N25G- 1.04 1.10 1.71 0.03 Y217Q-S24G- S53G-T55P-N25G-S53G- S87T-A88L- T55P-S87T- S89G-S101N- A88L-S89G- V203YS101N-V203Y FS1 G97A-G128A- N61P-S78N- 1.03 1.10 1.73 0.33 Y217Q-N61P-S101N-V203Y S78N-S101N- V203Y FS1 G97A-G128A- T55P-N61P- 1.06 1.10 1.970.39 Y217Q-T55P- S78N-S101N- N61P-S78N- V203Y S101N-V203Y FS1G97A-G128A- S53G-T55P- 1.03 1.10 2.22 0.33 Y217Q-S53G- N61P-S78N-T55P-N61P- S87T-A88L- S78N-S87T- S89G-S101N A88L-S89G- S101N RCL7G97A-G128A- V203Y-L267V 1.10 1.09 1.12 0.09 Y217Q-V203Y- L267V FS1G97A-G128A- S24G-N25G- 1.04 1.09 1.82 1.13 Y217Q-S24G- T55P-S101NN25G-T55P- S101N RCL7 G97A-G128A- A134T-L267V 1.13 1.08 0.93 0.66Y217Q-A134T- L267V FS1 G97A-G128A- S24G-N25G- 1.01 1.08 1.10 0.29Y217Q-S24G- S53G-T55P- N25G-S53G- N61P-S78N- T55P-N61P- S87T-A88L-S78N-S87T- S89G A88L-S89G FS1 G97A-G128A- S24G-N25G- 1.07 1.08 2.11 0.09Y217Q-S24G- S53G-N61P- N25G-S53G- S101N-V203Y N61P-S101N- V203Y RCL6G97A-G128A- N25Y-Q59S- 1.07 1.08 0.85 0.66 Y217Q-N25Y- N61P Q59S-N61PRCL7 G97A-G128A- I111V-S161P 1.10 1.08 0.66 0.98 Y217Q-I111V- S161P RCL7G97A-G128A- I115V-L267V 1.10 1.08 1.07 0.63 Y217Q-I115V- L267V FS1G97A-G128A- T55P-S78N- 0.99 1.08 1.39 0.16 Y217Q-T55P- S87T-A88L-S78N-S87T- S89G-S101N- A88L-S89G- V203Y S101N-V203Y RCL6 G97A-G128A-N25Y-P129Q- 1.07 1.08 0.88 0.67 Y217Q-N25Y- S130G-G131S- P129Q-S130G-A137T G131S-A137T RCL7 G97A-G128A- N61P-S63H- 1.04 1.08 1.59 0.78Y217Q-N61P- A128S-P129Q S63H-A128S- P129Q FS1 G97A-G128A- S53G-N61P-1.03 1.08 1.91 0.09 Y217Q-S53G- S101N-V203Y N61P-S101N- V203Y FS1G97A-G128A- S24G-N25G- 1.08 1.07 1.44 0.33 Y217Q-S24G- S53G-S78N-N25G-S53G- S87T-A88L- S78N-S87T- S89G-S101N A88L-S89G- S101N FS1G97A-G128A- N61P-S78N- 1.07 1.07 1.80 0.31 Y217Q-N61P- S87T-A88L-S78N-S87T- S89G-S101N A88L-S89G- S101N RCL6 G97A-G128A- N25Y-N61P- 1.061.07 0.74 0.60 Y217Q-N25Y- S63H N61P-S63H RCL5 G97A-G128A- Q59S-N61P-0.99 1.07 0.69 0.09 Y217Q-Q59S- V203Y N61P-V203Y RCL6 G97A-G128A-V8L-N25Y- 1.08 1.07 1.22 0.03 Y217Q-V8L- P129Q-S130G- N25Y-P129Q- G131SS130G-G131S RCL6 G97A-G128A- P86S-S87G- 1.16 1.07 1.26 0.03 Y217Q-P86S-A88V-P239R S87G-A88V- P239R FS1 G97A-G128A- S24G-N25G- 1.02 1.07 2.070.11 Y217Q-S24G- S53G-T55P- N25G-S53G- N61P-S101N- T55P-N61P- V203YS101N-V203Y RCL5 G97A-G128A- S24G-N25G- 1.09 1.06 1.25 1.00 Y217Q-S24G-P129Q-S130G- N25G-P129Q- G131S S130G-G131S FS3 G97A-G128A- N240K 1.041.06 0.90 0.94 Y217Q-N240K RCL5 G97A-G128A- G23A-S24G- 1.06 1.06 1.050.06 Y217Q-G23A- N25G-G211R- S24G-N25G- N212S-K213V G211R-N212S- K213VRCL7 G97A-G128A- N61P-S63H- 1.03 1.06 0.43 1.14 Y217Q-N61P- S78N-I111V-S63H-S78N- A134T I111V-A134T RCL6 G97A-G128A- S63T-P86S- 1.17 1.06 1.460.02 Y217Q-S63T- S87G-A88V P86S-S87G- A88V RCL6 G97A-G128A- G23A-S24G-1.08 1.06 0.91 0.21 Y217Q-G23A- N25G-A116N- S24G-N25G- N117S-N118GA116N-N117S- N118G FS1 G97A-G128A- S78N-S87T- 1.03 1.06 1.40 0.33Y217Q-S78N- A88L-S89G- S87T-A88L- S101N S89G-S101N RCL6 G97A-G128A-S24G-N25G- 1.06 1.06 0.74 1.05 Y217Q-S24G- A116N-N117S- N25G-A116N-N118G N117S-N118G RCL7 G97A-G128A- T55P-N240K 1.02 1.06 0.91 0.93Y217Q-T55P- N240K RCL6 G97A-G128A- T55P-P129V- 1.07 1.06 0.85 0.86Y217Q-T55P- P194S P129V-P194S RCL6 G97A-G128A- N25Y-S87G- 1.07 1.05 1.140.08 Y217Q-N25Y- A88V-S89A S87G-A88V- S89A FS1 G97A-G128A- S24G-N25G-1.02 1.05 1.19 0.06 Y217Q-S24G- S87T-A88L- N25G-S87T- S89G-S101NA88L-S89G- S101N RCL7 G97A-G128A- P129Q-S130G- 1.04 1.05 0.94 0.05Y217Q-P129Q- G131S-V203Y S130G-G131S- V203Y RCL6 G97A-G128A- Q59S-N61P-1.05 1.05 0.80 0.84 Y217Q-Q59S- N240K N61P-N240K FS3 G97A-G128A-S24R-P40E- 1.13 1.05 1.08 1.31 Y217Q-S24R- P129E-S159K- P40E-P129E-K265R S159K-K265R RCL7 G97A-G128A- P52S-T55P- 1.09 1.05 0.53 0.15Y217Q-P52S- V203Y T55P-V203Y RCL6 G97A-G128A- S24R-P129E 1.11 1.05 0.880.59 Y217Q-S24R- P129E FS1 G97A-G128A- S24G-N25G- 0.98 1.05 1.17 1.16Y217Q-S24G- S53G-N61P- N25G-S53G- S78N N61P-S78N FS1 G97A-G128A-S24G-N25G- 1.07 1.05 1.71 1.29 Y217Q-S24G- T55P-S78N- N25G-T55P- S101NS78N-S101N RCL6 G97A-G128A- P86S-S87G- 1.10 1.05 0.76 0.05 Y217Q-P86S-A88V-A116S- S87G-A88V- N117G-N118R A116S-N117G- N118R RCL6 G97A-G128A-N61P-S87T- 1.00 1.05 1.12 0.05 Y217Q-N61P- A88L-S89G S87T-A88L- S89G FS1G97A-G128A- S24G-N25G- 0.96 1.05 0.96 0.29 Y217Q-S24G- S53G-T55P-N25G-S53G- S78N-S87T- T55P-S78N- A88L-S89G S87T-A88L- S89G RCL5G97A-G128A- G23A-S24G- 1.06 1.05 0.89 0.13 Y217Q-G23A- N25G-N61P-S24G-N25G- S63H N61P-S63H RCL6 G97A-G128A- S24R-Q59S- 1.07 1.05 0.940.49 Y217Q-S24R- N61P Q59S-N61P RCL6 G97A-G128A- N61P-P129Q- 1.07 1.051.13 0.78 Y217Q-N61P- S130G-G131S P129Q-S130G- G131S FS1 G97A-G128A-S24G-N25G- 1.05 1.04 1.98 0.32 Y217Q-S24G- S53G-T55P- N25G-S53G-N61P-S78N- T55P-N61P- S87T-A88L- S78N-S87T- S89G-S101N A88L-S89G- S101NFS1 G97A-G128A- S24G-N25G- 1.05 1.04 1.57 0.15 Y217Q-S24G- S53G-T55P-N25G-S53G- S101N-V203Y T55P-S101N- V203Y FS1 G97A-G128A- N61P-S78N- 1.021.04 1.60 0.12 Y217Q-N61P- S87T-A88L- S78N-S87T- S89G-S101N- A88L-S89G-V203Y S101N-V203Y FS1 G97A-G128A- S24G-N25G- 1.03 1.04 2.01 1.27Y217Q-S24G- S53G-T55P- N25G-S53G- S78N-S101N T55P-S78N- S101N FS1G97A-G128A- S24G-N25G- 1.01 1.04 1.77 0.07 Y217Q-S24G- S53G-S101N-N25G-S53G- V203Y S101N-V203Y FS1 G97A-G128A- S24G-N25G- 1.09 1.04 1.540.28 Y217Q-S24G- S78N-S101N- N25G-S78N- V203Y S101N-V203Y RCL7G97A-G128A- P129Q-S130G- 1.14 1.04 1.37 0.56 Y217Q-P129Q- G131S-A133V-S130G-G131S- L267V A133V-L267V FS1 G97A-G128A- S87T-A88L- 1.05 1.04 1.440.07 Y217Q-S87T- S89G-S101N A88L-S89G- S101N RCL6 G97A-G128A- G23A-S24G-1.07 1.04 0.92 0.16 Y217Q-G23A- N25G-P239R S24G-N25G- P239R RCL6G97A-G128A- S87G-A88V- 1.07 1.04 0.92 0.37 Y217Q-S87G- S89A-A116N-A88V-S89A- N117S-N118G A116N-N117S- N118G RCL6 G97A-G128A- Q59S-N61P-1.09 1.04 0.68 0.91 Y217Q-Q59S- A116S-N117G- N61P-A116S- N118RN117G-N118R RCL5 G97A-G128A- Q59S-N61P- 1.03 1.04 0.81 0.08 Y217Q-Q59S-S87T-A88L- N61P-S87T- S89G A88L-S89G FS1 G97A-G128A- S24G-N25G- 0.961.04 0.73 0.04 Y217Q-S24G- S53G-S87T- N25G-S53G- A88L-S89G- S87T-A88L-V203Y S89G-V203Y RCL7 G97A-G128A- A134T-G211T 1.11 1.04 0.71 0.35Y217Q-A134T- G211T RCL7 G97A-G128A- T55P-A128S- 1.03 1.04 2.71 1.02Y217Q-T55P- P129Q A128S-P129Q FS1 G97A-G128A- T55P-S78N- 1.05 1.04 1.560.33 Y217Q-T55P- S87T-A88L- S78N-S87T- S89G-S101N A88L-S89G- S101N RCL6G97A-G128A- P86S-S87G- 1.09 1.04 0.83 0.04 Y217Q-P86S- A88V-T242RS87G-A88V- T242R RCL7 G97A-G128A- S161P-V203Y 1.04 1.04 0.81 0.09Y217Q-S161P- V203Y FS1 G97A-G128A- S24G-N25G- 1.05 1.04 2.01 0.44Y217Q-S24G- T55P-N61P- N25G-T55P- S78N-S101N- N61P-S78N- V203YS101N-V203Y RCL7 G97A-G128A- G211T-L267V 1.08 1.03 1.16 0.30Y217Q-G211T- L267V RCL6 G97A-G128A- P40E-T55P- 1.08 1.03 0.71 0.05Y217Q-P40E- N269K T55P-N269K RCL6 G97A-G128A- S24R-A128S- 1.08 1.03 1.630.64 Y217Q-S24R- P129G A128S-P129G RCL6 G97A-G128A- S24G-N25G- 1.15 1.033.06 0.35 Y217Q-S24G- N61P-N62S- N25G-N61P- P194L-A232T N62S-P194L-A232T RCL6 G97A-G128A- T55P-A116S- 1.07 1.03 0.83 1.06 Y217Q-T55P-N117G-N118R A116S-N117G- N118R FS1 G97A-G128A- S24G-N25G- 1.03 1.03 1.760.32 Y217Q-S24G- S53G-S78N- N25G-S53G- S101N-V203Y S78N-S101N- V203YRCL7 G97A-G128A- P129Q-S130G- 1.08 1.03 0.90 0.83 Y217Q-P129Q-G131S-N240K S130G-G131S- N240K FS1 G97A-G128A- S53G-T55P- 0.93 1.03 1.000.33 Y217Q-S53G- N61P-S78N- T55P-N61P- S87T-A88L- S78N-S87T- S89GA88L-S89G RCL6 G97A-G128A- N25Y-P129Q- 1.08 1.03 0.93 0.69 Y217Q-N25Y-S130G-G131S P129Q-S130G- G131S RCL6 G97A-G128A- T55P-I115V 1.06 1.031.07 1.07 Y217Q-T55P- I115V RCL6 G97A-G128A- N25Y-T55P 1.05 1.03 1.000.80 Y217Q-N25Y- T55P RCL6 G97A-G128A- G23A-S24G- 0.97 1.03 1.75 0.15Y217Q-G23A- N25G-A128S- S24G-N25G- P129D A128S-P129D FS1 G97A-G128A-S53G-S78N- 1.00 1.03 1.68 0.13 Y217Q-S53G- S87T-A88L- S78N-S87T-S89G-S101N- A88L-S89G- P129S-V203Y S101N-P129S- V203Y RCL7 G97A-G128A-T55P-A134T 1.06 1.03 0.94 1.05 Y217Q-T55P- A134T RCL7 G97A-G128A-N61P-S63H- 1.03 1.02 0.49 1.18 Y217Q-N61P- S78N-I111V S63H-S78N- I111VFS2 Y217Q-N61P- N61P-A97G- 0.98 1.02 NA NA A97G-G102A- G102A-A128G-A128G-P129S P129S FS1 G97A-G128A- S53G-N61P- 1.02 1.02 2.08 0.88Y217Q-S53G- S101N N61P-S101N RCL5 G97A-G128A- Q59S-N61P- 1.01 1.02 0.900.20 Y217Q-Q59S- S87G-A88V- N61P-S87G- S89A A88V-S89A FS1 G97A-G128A-S53G-S87T- 0.96 1.02 1.11 0.04 Y217Q-S53G- A88L-S89G- S87T-A88L-S101N-V203Y S89G-S101N- V203Y RCL6 G97A-G128A- S87T-A88L- 1.06 1.02 1.210.10 Y217Q-S87T- S89G-P129S A88L-S89G- P129S FS1 G97A-G128A- S53G-T55P-1.03 1.02 1.70 0.48 Y217Q-S53G- S78N-S101N- T55P-S78N- V203Y S101N-V203YRCL5 G97A-G128A- T55P-P129Q- 1.01 1.02 1.18 0.91 Y217Q-T55P- S130G-G131SP129Q-S130G- G131S RCL5 G97A-G128A- Q59S-N61P- 0.98 1.02 0.83 0.80Y217Q-Q59S- P129Q-S130G- N61P-P129Q- G131S S130G-G131S RCL7 G97A-G128A-A134T-P239R 1.03 1.02 0.53 0.98 Y217Q-A134T- P239R RCL5 G97A-G128A-T55P-V203Y 1.01 1.01 1.02 0.23 Y217Q-T55P- V203Y RCL7 G97A-G128A-T55P-S78N- 1.03 1.01 1.05 1.25 Y217Q-T55P- S89Y S78N-S89Y RCL5G97A-G128A- T22N-S24A- 1.00 1.01 0.69 0.46 Y217Q-T22N- N61P-S63HS24A-N61P- S63H RCL7 G97A-G128A- S161P-L267V 1.05 1.01 1.00 0.66Y217Q-S161P- L267V RCL6 G97A-G128A- T55P-L75H- 1.06 1.01 0.69 0.59Y217Q-T55P- N76G L75H-N76G RCL7 G97A-G128A- A134T-S161P 1.07 1.01 0.731.00 Y217Q-A134T- S161P RCL7 G97A-G128A- S87T-A88L- 1.08 1.01 0.66 0.13Y217Q-S87T- S89G-A134T A88L-S89G- A134T RCL6 G97A-G128A- T55P-A116N-1.06 1.01 1.06 1.11 Y217Q-T55P- N117S-N118G A116N-N117S- N118G v12G97A-G128S- A128S 1.02 1.01 1.65 1.00 Y217Q RCL7 G97A-G128A- T55P-S78N-1.07 1.00 1.00 1.32 Y217Q-T55P- I115V S78N-I115V RCL6 G97A-G128A-Y6Q-P129Q- 1.03 1.00 0.98 0.23 Y217Q-Y6Q- S130G-G131S P129Q-S130G- G131SRCL7 G97A-G128A- S24R-P129Q- 1.06 1.00 1.11 0.61 Y217Q-S24R- S130G-G131SP129Q-S130G- G131S FS1 G97A-G128A- S24G-N25G- 0.99 1.00 1.61 1.21Y217Q-S24G- S53G-S78N- N25G-S53G- S101N S78N-S101N RCL6 G97A-G128A-T55P-P129V 1.07 1.00 0.70 1.00 Y217Q-T55P- P129V v3 G97A-G128A- BPN′-v31.00 1.00 1.00 1.00 Y217Q FS2 G97A-Y217Q- N61P-N62Q- 0.95 1.00 NA NAN61P-N62Q- G100N-A128G G100N-A128G RCL6 G97A-G128A- T55P-P129Q 1.10 1.001.77 1.15 Y217Q-T55P- P129Q FS1 G97A-G128A- S24G-N25G- 1.05 1.00 1.840.12 Y217Q-S24G- S53G-T55P- N25G-S53G- N61P-S78N- T55P-N61P- S87T-A88L-S78N-S87T- S89G-S101N- A88L-S89G- V203Y S101N-V203Y RCL6 G97A-G128A-S87T-A88L- 1.04 1.00 0.83 0.10 Y217Q-S87T- S89G-N240K A88L-S89G- N240KRCL7 G97A-G128A- A134T-N240K 1.07 0.99 0.61 0.96 Y217Q-A134T- N240K RCL7G97A-G128A- S87T-A88L- 1.04 0.99 0.68 0.14 Y217Q-S87T- S89G-P239RA88L-S89G- P239R RCL7 G97A-G128A- P129Q-S130G- 1.08 0.99 1.35 0.61Y217Q-P129Q- G131S-L267V S130G-G131S- L267V RCL7 G97A-G128A- P129Q-N240K1.06 0.99 1.39 1.00 Y217Q-P129Q- N240K FS1 G97A-G128A- S78N-S87T- 0.930.99 0.74 0.15 Y217Q-S78N- A88L-S89G- S87T-A88L- V203Y S89G-V203Y RCL7G97A-G128A- I111V-A273S 1.00 0.99 0.48 0.27 Y217Q-I111V- A273S FS1G97A-G128A- S24G-N25G- 1.03 0.99 2.17 0.71 Y217Q-S24G- T55P-S78N-N25G-T55P- A88V-S101N S78N-A88V- S101N FS1 G97A-G128A- S24G-N25G- 0.960.98 1.15 1.20 Y217Q-S24G- T55P-S78N N25G-T55P- S78N FS1 G97A-G128A-S24G-N25G- 1.02 0.98 1.27 0.10 Y217Q-S24G- S53G-S78N- N25G-S53G-S87T-A88L- S78N-S87T- S101N-V203Y A88L-S101N- V203Y FS1 G97A-G128A-S24G-N25G- 0.97 0.98 0.75 0.14 Y217Q-S24G- S53G-S78N- N25G-S53G-S87T-A88L- S78N-S87T- S89G-V203Y A88L-S89G- V203Y FS1 G97A-G128A-S24G-N25G- 1.06 0.98 1.21 0.14 Y217Q-S24G- S53G-S78N- N25G-S53G-S87T-A88L- S78N-S87T- S89G-S101N- A88L-S89G- V203Y S101N-V203Y RCL5G97A-G128A- S87G-A88V- 1.01 0.98 1.18 0.29 Y217Q-S87G- S89A-P129Q-A88V-S89A- S130G-G131S P129Q-S130G- G131S RCL7 G97A-G128A- N61P-S63H-1.03 0.98 0.73 1.16 Y217Q-N61P- S78N-S161P S63H-S78N- S161P FS1G97A-G128A- T55P-N61P- 1.04 0.98 1.75 0.14 Y217Q-T55P- S78N-S87T-N61P-S78N- A88L-S89G- S87T-A88L- S101N-V203Y S89G-S101N- V203Y RCL7G97A-G128A- I111V-P129Q- 1.00 0.98 0.52 0.97 Y217Q-I111V- S130G-G131SP129Q-S130G- G131S RCL5 G97A-G128A- T22N-S24A- 0.96 0.98 0.94 0.64Y217Q-T22N- T55P S24A-T55P RCL7 G97A-G128A- I115V-N240K 1.04 0.98 0.660.90 Y217Q-I115V- N240K RCL6 G97A-G128A- S87G-A88V- 0.98 0.98 0.66 0.40Y217Q-S87G- S89A-A116N- A88V-S89A- N117S-N118G- A116N-N117S- P172HN118G-P172H FS1 G97A-G128A- S24G-N25G- 1.01 0.98 1.28 0.31 Y217Q-S24G-S78N-S87T- N25G-S78N- A88L-S89G- S87T-A88L- S101N S89G-S101N RCL6G97A-G128A- S24G-N25G- 1.03 0.98 0.59 1.13 Y217Q-S24G- I115V-A134TN25G-I115V- A134T RCL6 G97A-G128A- T55P-A128S- 0.97 0.97 1.39 1.00Y217Q-T55P- P129D A128S-P129D RCL7 G97A-G128A- I111V-S159K 0.99 0.970.64 1.12 Y217Q-I111V- S159K RCL7 G97A-G128A- N240K-A273S 1.03 0.96 0.640.19 Y217Q-N240K- A273S RCL7 G97A-G128A- S159K-L267V 1.00 0.96 1.08 0.79Y217Q-S159K- L267V RCL7 G97A-G128A- I111V-P129Q- 1.01 0.96 0.83 0.42Y217Q-I111V- G211T P129Q-G211T RCL7 G97A-G128A- I115V-A273S 1.05 0.950.74 0.20 Y217Q-I115V- A273S RCL6 G97A-G128A- S89Y 0.99 0.95 0.70 0.88Y217Q-S89Y RCL6 G97A-G128A- S24R-A116N- 1.08 0.95 0.91 0.60 Y217Q-S24R-N117S-N118G A116N-N117S- N118G RCL7 G97A-G128A- N61E-A144K 1.03 0.950.97 1.10 Y217Q-N61E- A144K RCL6 G97A-G128A- P129Q-S130G- 1.02 0.95 0.850.99 Y217Q-P129Q- G131S-P239R S130G-G131S- P239R RCL7 G97A-G128A-S87T-A88L- 0.97 0.95 0.59 0.13 Y217Q-S87T- S89G-I115V A88L-S89G- I115VRCL6 G97A-G128A- T55P-A92G 0.96 0.94 0.56 0.91 Y217Q-T55P- A92G FS3G97A-G128A- S145D-S159K- 0.98 0.94 0.76 1.08 Y217Q-S145D- N240K-Q275ES159K-N240K- Q275E RCL7 G97A-G128A- S89Y-P129Q- 1.04 0.94 0.71 0.70Y217Q-S89Y- S130G-G131S P129Q-S130G- G131S RCL7 G97A-G128A- P129Q-S130G-1.01 0.94 1.00 0.93 Y217Q-P129Q- G131S-S162K S130G-G131S- S162K RCL7G97A-G128A- I111V-A134T 0.98 0.94 0.37 1.06 Y217Q-I111V- A134T RCL6G97A-G128A- P40E-S53Y- 0.99 0.94 0.97 0.35 Y217Q-P40E- S78Y-P86S-S53Y-S78Y- S87G-A88V P86S-S87G- A88V RCL6 G97A-G128A- S24G-N25G- 0.930.93 0.58 0.52 Y217Q-S24G- L75H-N76G N25G-L75H- N76G FS2 G97A-Y217Q-N61P-A128G- 0.85 0.93 0.65 0.92 N61P-A128G- P129S-S130P P129S-S130P RCL6G97A-G128A- S24R-S145D 0.99 0.93 0.89 0.59 Y217Q-S24R- S145D FS3G97A-G128A- S24R-S145D- 0.92 0.92 0.63 0.68 Y217Q-S24R- P239R-Q275ES145D-P239R- Q275E RCL7 G97A-G128A- S24R-S78N- 0.95 0.92 1.16 0.55Y217Q-S24R- S182P-L267V S78N-S182P- L267V FS1 G97A-G128A- S53G-N61P-1.04 0.92 1.67 0.02 Y217Q-S53G- S87T-A88L- N61P-S87T- S89G-S101N-A88L-S89G- V203Y S101N-V203Y RCL6 G97A-G128A- P5S-S87G- 0.98 0.92 0.640.07 Y217Q-P5S- A88V-S89A- S87G-A88V- A116G-N117R S89A-A116G- N117R FS1G97A-G128A- S53G-N61P- 0.99 0.92 1.65 0.11 Y217Q-S53G- S78N-S87T-N61P-S78N- A88L-S89G- S87T-A88L- S101N-V203Y S89G-S101N- V203Y RCL6G97A-G128A- Q59S-N61P- 0.93 0.92 0.42 1.04 Y217Q-Q59S- A116N-N117S-N61P-A116N- N118G N117S-N118G RCL7 G97A-G128A- P239R-A273S 0.94 0.910.52 0.23 Y217Q-P239R- A273S FS1 G97A-G128A- S53G-S78N- 0.94 0.91 1.160.13 Y217Q-S53G- S87T-A88L- S78N-S87T- S89G-S101N- A88L-S89G- V203YS101N-V203Y RCL6 G97A-G128A- S24R-P129V 0.98 0.91 0.52 0.58 Y217Q-S24R-P129V RCL7 G97A-G128A- I111V-P239R 0.97 0.91 0.38 1.08 Y217Q-I111V-P239R FS1 G97A-G128A- S87T-A88L- 0.90 0.91 0.79 0.05 Y217Q-S87T-S89G-S101N- A88L-S89G- V203Y S101N-V203Y RCL6 G97A-G128A- T55P-P129L0.99 0.91 0.62 0.98 Y217Q-T55P- P129L RCL7 G97A-G128A- S87T-A88L- 0.920.90 0.42 0.13 Y217Q-S87T- S89G-I111V A88L-S89G- I111V RCL7 G97A-G128A-S145D-A273S 0.91 0.90 0.66 0.17 Y217Q-S145D- A273S RCL6 G97A-G128A-P129Q-S130G- 0.94 0.90 0.51 0.89 Y217Q-P129Q- G131S-T242R S130G-G131S-T242R RCL7 G97A-G128A- S3F-S87T- 0.93 0.89 0.55 0.07 Y217Q-S3F-A88L-S89G- S87T-A88L- G211T S89G-G211T RCL6 G97A-G128A- S87G-A88V- 0.970.89 1.21 0.37 Y217Q-S87G- S89A-S162K A88V-S89A- S162K RCL7 G97A-G128A-S89Y-G211T 0.89 0.88 0.53 0.41 Y217Q-S89Y- G211T RCL7 G97A-G128A-S87T-A88L- 0.93 0.88 0.74 0.12 Y217Q-S87T- S89G-A144K A88L-S89G- A144KRCL6 G97A-G128A- P129Q-S130G- 0.95 0.88 1.21 0.96 Y217Q-P129Q-G131S-S159K S130G-G131S- S159K RCL6 G97A-G128A- A116N-N117S- 0.90 0.880.54 0.95 Y217Q-A116N- N118G-P129Q- N117S-N118G- S130G-G131SP129Q-S130G- G131S RCL6 G97A-G128A- S24G-N25G- 0.86 0.87 0.44 1.02Y217Q-S24G- P129V N25G-P129V FS1 G97A-G128A- S24G-N25G- 0.87 0.86 0.830.12 Y217Q-S24G- S78N-S87T- N25G-S78N- A88L-S89G- S87T-A88L- S101N-V203YS89G-S101N- V203Y FS2 G97A-Y217Q- N123G-A128G 0.83 0.86 1.00 0.14N123G-A128G FS2 G97A-G128A- N61P-N62Q- 0.82 0.86 5.52 0.40 Y217Q-N61P-G100N-G102A- N62Q-G100N- M124I G102A-M124I RCL6 G97A-G128A- S24G-N25G-0.90 0.85 0.65 1.10 Y217Q-S24G- K141E-T242R N25G-K141E- T242R RCL6G97A-G128A- S87G-A88V- 0.89 0.85 0.44 0.32 Y217Q-S87G- S89A-A116N-A88V-S89A- N117S-N118G- A116N-N117S- A144T N118G-A144T FS1 G97A-G128A-T55P-N61P- 0.83 0.85 0.92 0.30 Y217Q-T55P- S87T-A88L- N61P-S87T-S89G-G110C- A88L-S89G- S130P G110C-S130P RCL6 G97A-G128A- L75S-N76Y-0.84 0.83 0.41 0.13 Y217Q-L75S- A116S-N117G- N76Y-A116S- N118RN117G-N118R FS3 G97A-G128A- S145D-S159K- 0.74 0.81 0.38 0.65Y217Q-S145D- K213L-P239R- S159K-K213L- N240K P239R-N240K RCL6G97A-G128A- S24R-S87T- 0.86 0.80 0.53 0.08 Y217Q-S24R- A88L-S89GS87T-A88L- S89G RCL6 G97A-G128A- G23A-S24G- 0.88 0.79 0.53 0.20Y217Q-G23A- N25G-P129V S24G-N25G- P129V RCL7 G97A-G128A- A134T-K213L0.87 0.79 0.51 0.81 Y217Q-A134T- K213L RCL7 G97A-G128A- S89Y-A273S 0.810.79 0.48 0.19 Y217Q-S89Y- A273S RCL7 G97A-G128A- S24R-P239R 0.90 0.780.71 0.69 Y217Q-S24R- P239R FS2 G97A-Y217Q- N123G-A128G- 0.76 0.78 0.710.12 N123G-A128G- P129S P129S RCL7 G97A-G128A- S89Y-P239R 0.77 0.76 0.380.90 Y217Q-S89Y- P239R RCL6 G97A-G128A- S24G-N25G- 0.71 0.76 0.25 0.59Y217Q-S24G- A92G N25G-A92G RCL6 G97A-G128A- N61P-S63H- 0.73 0.74 0.240.42 Y217Q-N61P- I115V-A228V S63H-I115V- A228V FNA Y217L A97G-A128G-0.73 0.74 1.81 1.21 Q217L RCL6 G97A-G128A- L75S-N76Y- 0.73 0.73 0.340.09 Y217Q-L75S- P129V N76Y-P129V RCL6 G97A-G128A- S24R-P129L 0.80 0.730.35 0.58 Y217Q-S24R- P129L RCL6 G97A-G128A- S87G-A88V- 0.69 0.73 0.900.36 Y217Q-S87G- S89A-P129Q- A88V-S89A- S182Y-S204Y- P129Q-S182Y- P239QS204Y-P239Q RCL6 G97A-G128A- S24R-A92G 0.75 0.73 0.28 0.44 Y217Q-S24R-A92G RCL6 G97A-G128A- S24R-A116S- 0.81 0.72 0.48 0.58 Y217Q-S24R-N117G-N118R A116S-N117G- N118R RCL6 G97A-G128A- G23A-S24G- 0.85 0.660.63 0.20 Y217Q-G23A- N25G-A116G- S24G-N25G- N117R A116G-N117R RCL6G97A-G128A- S24G-N25G- 0.72 0.66 0.39 1.03 Y217Q-S24G- P129L N25G-P129LRCL6 G97A-G128A- S87T-A88L- 0.78 0.66 0.65 0.11 Y217Q-S87T- S89G-S101GA88L-S89G- S101G RCL6 G97A-G128A- G23A-S24G- 0.52 0.59 0.37 0.20Y217Q-G23A- N25G-P129L S24G-N25G- P129L FS1 G97A-G128A- S53G-N61P- 0.550.58 0.29 0.16 Y217Q-S53G- G102A-V203Y N61P-G102A- V203Y RCL6G97A-G128A- T55P-V147P 0.56 0.57 0.22 1.09 Y217Q-T55P- V147P RCL6G97A-G128A- Y6Q-L75S- 0.55 0.52 0.24 0.17 Y217Q-Y6Q- N76Y L75S-N76Y RCL6G97A-G128A- N61P-S63H- 0.36 0.37 0.14 0.95 Y217Q-N61P- V147P S63H-V147PRCL6 G97A-G128A- S24R-V147P 0.16 0.22 0.08 0.85 Y217Q-S24R- V147P RCL6G97A-G128A- S24G-N25G- 0.21 0.18 0.06 0.71 Y217Q-S24G- V68C-A69GN25G-V68C- A69G FS1 G97A-G128A- S24G-N25G- 0.19 0.18 0.08 0.38Y217Q-S24G- N61P-L75S- N25G-N61P- N76Y-S101N- L75S-N76Y- V203YS101N-V203Y FS1 G97A-G128A- L75S-N76Y- 0.13 0.12 0.05 0.72 Y217Q-L75S-S78N-S101N- N76Y-S78N- V203Y S101N-V203Y FS1 G97A-G128A- L75S-N76Y- 0.040.04 0.07 0.73 Y217Q-L75S- S78N-S87T- N76Y-S78N- A88L-S89G- S87T-A88L-S101N-S130P S89G-S101N- S130P FS1 G97A-G128A- S24G-N25G- 0.06 0.04 0.060.33 Y217Q-S24G- S53G-S101N- N25G-S53G- S130P-V203Y S101N-S130P- V203YRCL6 G97A-G128A- G47E-M50I-

0.03 0.02 1.21 Y217Q-G47E- L75S-N76Y- M50I-L75S- S162K N76Y-S162K FS1G97A-G128A- S53G-T55P-

0.02 0.05 0.45 Y217Q-S53G- S87T-A88L- T55P-S87T- S89G-S101N- A88L-S89G-S130P-V203Y S101N-S130P- V203Y FS1 G97A-G128A- S24G-N25G-

0.02 1.21 Y217Q-S24G- L75S-N76Y- N25G-L75S- A128T-P129T- N76Y-A128T-S130G-G131Q- P129T-S130G- S132C-A133G- G131Q-S132C- A134T A133G-A134TRCL6 G97A-G128A- P129Q-S130G-

0.03 1.11 Y217Q-P129Q- G131S-V147P S130G-G131S- V147P FS1 G97A-G128A-S53G-T55P-

0.02 1.05 Y217Q-S53G- N61P-L75S- T55P-N61P- N76Y-S87T- L75S-N76Y-A88L-S89G- S87T-A88L- G102A-S130P- S89G-G102A- V203Y S130P-V203Y FS1G97A-G128A- S53G-T55P- 0.03

0.02 1.03 Y217Q-S53G- N61P-L75S- T55P-N61P- N76Y-S101N- L75S-N76Y-S130P-V203Y S101N-S130P- V203Y

The invention includes a protease variant having proteolytic activity,the variant comprising an amino acid sequence having at least 60%, 70%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identityto SEQ ID NO:2 or SEQ ID NO:6 and at least one set of amino acidsubstitutions selected from those listed in Table 12-4, whereinpositions of the variant are numbered by correspondence with positionsof the SEQ ID NO:2 sequence. Each such protease variant may be anisolated, recombinant, substantially pure, or non-naturally occurringprotease variant. Also included are compositions, including cleaningcompositions, comprising at least one such protease variant and methodsfor cleaning utilizing at least one such protease variant as describedin greater detail elsewhere herein.

Table 12-5 provides the Performance Index (PI) values of BPN′ variants(generated as described in “Generation of Variants to Improve BPN′Stability”; see Table 11-3) for stain removal in BMI microswatch assayin Detergent Composition 1 at 16° C. and pH 8 (Det. Comp. 1, pH 8, 16°C., BMI PI) and for stability in LAS/EDTA (LAS/EDTA Stability PI).Assays were performed as described in Example 1 (BMI microswatch assay,LAS/EDTA stability assay). The sequences of the variants are shownrelative to both BPN′ and FNA. That is, each variant sequence is theBPN′ or FNA sequence with the specified variant amino acidsubstitutions. PI values are shown relative to FNA parent, which isBPN′-Y217L.

TABLE 12-5 Performance Index of Stability-Improved BPN′ VariantsSequence Relative to Det. Comp. LAS/EDTA FNA: BPN′ Sequence 1 pH 8, 16°Stability Y217L Relative to BPN′ C., BMI PI PI P40E-S78N-P40E-S78N-S87D- 0.71 11.65 S87D Y217L P40E P40E-Y217L 0.96 8.33T22V-S78N- T22V-S78N-Q206E- 0.75 5.95 Q206E-K213N K213N-Y217L T22V-S78N-T22V-S78N-K213N- 0.86 5.71 K213N Y217L S87D S87D-Y217L 0.90 4.04 S78NS78N-Y217L 0.87 3.86 K213N K213N-Y217L 0.91 1.84 Q206E Q206E-Y217L 0.861.76 T22V T22V-Y217L 0.97 1.46 FNA Y217L 1.00 1.00

The invention includes a protease variant having proteolytic activityand/or improved stability relative to FNA, the variant comprising anamino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ IDNO:6 and at least one set of amino acid substitutions selected fromthose listed in Table 12-5, wherein positions of the variant arenumbered by correspondence with positions of the SEQ ID NO:2 sequence.Each such protease variant may be an isolated, recombinant,substantially pure, or non-naturally occurring protease variant. Alsoincluded are compositions, including cleaning compositions, comprisingat least one such protease variant and methods for cleaning utilizing atleast one such protease variant as described in greater detail elsewhereherein.

Table 12-6 provides the Performance Index (PI) values of BPN′ variantsgenerated from Library Parent: BPN′-v3: G97A-G128A-Y217Q (as describedin “Generation of Variants to Improve BPN′ Stability”; see Table 11-3)for stain removal in a BMI microswatch assay in Detergent Composition 1at 16° C. and pH 8 and for stability in LAS/EDTA. Assays were performedas described in Example 1 (BMI microswatch assay and LAS/EDTA stabilityassay). The Performance Index was calculated relative to BPN′-v3:G97A-G128A-Y217Q. All mutants in this list have a PI cutoff equal orgreater than 0.5 for at least one property tested.

TABLE 12-6 Performance Index of Stability-Improved BPN′ VariantsSequence Det. Comp. LAS/ Relative to 1 pH 8, EDTA BPN′-v3: BPN′ Sequence16° C., Stability G97A-G128A-Y217Q Relative to BPN′ BMI PI PIS87D-N76D-S78N S87D-N76D-S78N- 0.62 2.27 G97A-G128A-Y217Q P40E-S78N-S87DP40E-S78N-S87D- 0.21 2.18 G97A-G128A-Y217Q P40E-S87D P40E-S87D-G97A-0.18 2.14 G128A-Y217Q S78N-P40E S78N-P40E-G97A- 0.80 2.03 G128A-Y217QS87D-N76D S87D-N76D-G97A- 0.55 1.89 G128A-Y217Q P40E P40E-G97A-G128A-0.84 1.79 Y217Q S78N-S87D S78N-S87D-G97A- 0.79 1.71 G128A-Y217Q S87DS87D-G97A-G128A- 0.78 1.20 Y217Q S78N S78N-G97A-G128A- 0.93 1.14 Y217QBPN′-v3 G97A-G128A-Y217Q 1.00 1.00

The invention includes a protease variant having proteolytic activityand having improved stability relative to BPN′-v3, the variantcomprising an amino acid sequence having at least 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ IDNO:2 or SEQ ID NO:6 and at least one set of amino acid substitutionsselected from those listed in Table 12-6, wherein positions of thevariant are numbered by correspondence with positions of the SEQ ID NO:2sequence. Each such protease variant may be an isolated, recombinant,substantially pure, or non-naturally occurring protease variant. Alsoincluded are compositions, including cleaning compositions, comprisingat least one such protease variant and methods for cleaning utilizing atleast one such protease variant as described in greater detail elsewhereherein.

Table 12-7 provides the Performance Index (PI) values of BPN′ variants(generated as described in “Generation of BPN′ Variants from FiveDifferent Plasmids”; see Table 11-4) for stain removal in a BMImicroswatch assay in Detergent Composition lat 16° C. and pH 8. Assayswere performed as described in Example 1 (BMI microswatch assay). ThePerformance Index of each variant was calculated relative to BPN′-v3:G97A-G128A-Y217Q. All mutants in this list have a PI cutoff equal orgreater than 0.5 for at least one property tested.

TABLE 12-7 Performance Index of BPN′ Variants Sequence Relative to Det.Comp. 1, BPN′-v3 G97A- pH 8, 16° G128A-Y217Q Sequence Relative to BPN′C., BMI PI S101N G97A-S101N-G128A-Y217Q 1.12 A137VG97A-G128A-A137V-Y217Q 1.12 N61P N61P-G97A-G128A-Y217Q 1.11 S130PG97A-G128A-S130P-Y217Q 1.09 Q103N G97A-Q103N-G128A-Y217Q 1.07 S63TS63T-G97A-G128A-Y217Q 1.03 G102A G97A-G102A-G128A-Y217Q 1.02 BPN′-v3BPN′-v3 (G97A-G128A-Y217Q) 1.00 N109D-S248R G97A-N109D-G128A-Y217Q-S248R0.96 S87R S87R-G97A-G128A-Y217Q 0.95 S188D G97A-G128A-S188D-Y217Q 0.95S87D-S248R S87D-G97A-G128A-Y217Q-S248R 0.94 S188D-S248RG97A-G128A-S188D-S248R-Y217Q 0.93 S248D G97A-G128A-S248D-Y217Q 0.86N109D-S188D- G97A-N109D-G128A-S188D- 0.83 S248R S248R-Y217Q N109DG97A-N109D-G128A-Y217Q 0.81 S87R-S248R S87R-G97A-G128A-Y217Q-S248R 0.79N109D-S188R G97A-N109D-G128A-S188R- 0.77 Y217Q N76DN76D-G97A-G128A-Y217Q 0.75 S87D-N109D- S87D-G97A-N109D-G128A- 0.58S188D-S248R S188D-S248R-Y217Q S87R-N109D- S87R-G97A-N109D-G128A- 0.55S188D-S248R S188D-Y217Q-S248R S87R-S188R- S87R-G97A-G128A-S188R- 0.52S248R Y217Q-S248R A187D G97A-G128A-A187D-Y217Q 0.48 N109D-S248DG97A-N109D-G128A-Y217Q- 0.47 S248D S87R-N109R- S87R-G97A-N109R-G128A-0.39 S188R-S248R S188R-Y217Q-S248R F189D G97A-G128A-F189D-Y217Q 0.31G100N G97A-G100N-G128A-Y217Q 0.28 S87R-N109D- S87R-G97A-N109D-G128A-0.24 S188D S188D-Y217Q S87D-N109D- S87D-G97A-N109D-G128A- 0.12 S188DS188D-Y217Q S87R-S188D- S87R-G97A-G128A-S188D- 0.09 S248D S248D-Y217QN62D N62D-G97A-G128A-Y217Q 0.09 S87D-N109D- S87D-G97A-N109D-G128A- 0.08S188D-S248D S188D-Y217Q-S248D

The invention includes a protease variant having proteolytic activity,the variant comprising an amino acid sequence having at least 60%, 70%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identityto SEQ ID NO:2 or SEQ ID NO:6 and at least one set of amino acidsubstitutions selected from those listed in Table 12-7, whereinpositions of the variant are numbered by correspondence with positionsof the SEQ ID NO:2 sequence. Each such protease variant may be anisolated, recombinant, substantially pure, or non-naturally occurringprotease variant. Also included are compositions, including cleaningcompositions, comprising at least one such protease variant and methodsfor cleaning utilizing at least one such protease variant as describedin greater detail elsewhere herein.

Table 12-8 provides the Performance index (PI) values of BPN′ variants(generated as described in “Generation of Combinatorial Libraries andVariants of BPN′-v3+578N” as described in Example 3) for stain removalusing a BMI microswatch assay in Detergent Composition 1 at 16° C. andpH 8. Assays were performed as described in Example 1 (BMI microswatchassay). The Performance Index of each variant was calculated relative toBPN′-S78N-G97A-G128A-Y217Q. PI values less than 0.01 were modified andare indicated as “0.01” in bold italics.

TABLE 12-8 Performance Index Values of BPN′ Variants Sequence Det.Relative to Comp. BPN′-v3: 1 pH 8, Sequence G97A- 16° C., VariantRelative to BPN′ G128A-Y217Q BMI PI v3/S78N/L267V S78N-G97A-G128A-S78N-L267V 1.12 Y217Q-L267V v3/S78N/S161P S78N-G97A-G128A- S78N-S161P1.05 Y217Q-S161P v3/S78N/I115V S78N-G97A-G128A- S78N-I115V 1.04Y217Q-I115V v3/S78N/A273S S78N-G97A-G128A- S78N-A273S 1.03 Y217Q-A273Sv3/S78N/G211T S78N-G97A-G128A- S78N-G211T 1.00 Y217Q-G211T V3 + S78NS78N-G97A-G128A- S78N 1.00 Y217Q v3/S78N/I111V S78N-G97A-G128A-S78N-I111V 0.98 Y217Q-I111V v3/S78N/V147L S78N-G97A-G128A- S78N-V147L0.97 Y217Q-V147L v3/S78N/I108V S78N-G97A-G128A- S78N-I108V 0.97Y217Q-I108V v3/S78N/S89Y S78N-G97A-G128A- S78N-S89Y 0.94 Y217Q-S89Yv3/S78N/A138T S78N-G97A-G128A- S78N-A138T 0.92 Y217Q-A138T v3/S78N/P172VS78N-G97A-G128A- S78N-P172V 0.74 Y217Q-P172V v3/S78N/Q59GS78N-G97A-G128A- S78N-Q59G 0.64 Y217Q-Q59G GcM96 G97A-G128A-Y217Q-P129T-V147Q- 0.57 P129T-V147Q-S159D- S159D-S161P- S161P-S183T-Q185T-S183T-Q185T- G211A-S224A G211A-S224A GcM91 G97A-G128A-Y217Q-Q059V-I108V- 0.55 Q059V-I108V-V147Q- V147Q-G211A- G211A-N252Q N252Qv3/S78N/Y167A S78N-G97A-G128A- S78N-Y167A 0.53 Y217Q-Y167A v3/S78N/A92GS78N-G97A-G128A- S78N-A92G 0.49 Y217Q-A92G v3/S78N/P129LS78N-G97A-G128A- S78N-P129L 0.48 Y217Q-P129L GcM92 G97A-G128A-Y217Q-N061A-S087E- 0.36 N061A-S087E-M124I- M124I-S161P- S161P-S224A S224Av3/S78N/N62Q S78N-G97A-G128A- S78N-N62Q 0.27 Y217Q-N62Q v3/S78N/V68AS78N-G97A-G128A- S78N-V68A 0.24 Y217Q-V68A GcM94 G97A-G128A-Y217Q-S063T-S101A- 0.12 S063T-S101A-L126V- L126V-S183T- S183T-T244N T244Nv3/S78N/M124T S78N-G97A-G128A- S78N-M124T 0.05 Y217Q-M124T GcM95G97A-G128A-Y217Q- P040L-S053G-

P040L-S053G-Q059V- Q059V-N061A- N061A-N062Q-S063T- N062Q-S063T-S087E-G100N S087E-G100N GcM93 G97A-G128A-Y217Q- N062Q-G100N-

N062Q-G100N-S125A- S125A-S159D- S159D-N240S N240S GcM100G97A-G128A-Y217Q- V68A-G102A-

V68A-G102A-G211A- G211A-S125A S125A GcM90 G97A-G128A-Y217Q- S053G-V068A-

S053G-V068A-G102A- G102A-P129T- P129T-Q185T Q185T

The invention includes a protease variant having proteolytic activity,the variant comprising an amino acid sequence having at least 60%, 70%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identityto SEQ ID NO:2 or SEQ ID NO:6 and at least one set of amino acidsubstitutions selected from those listed in Table 12-8, whereinpositions of the variant are numbered by correspondence with positionsof the SEQ ID NO:2 sequence. Each such protease variant may be anisolated, recombinant, substantially pure, or non-naturally occurringprotease variant. Also included are compositions, including cleaningcompositions, comprising at least one such protease variant and methodsfor cleaning utilizing at least one such protease variant as describedin greater detail elsewhere herein.

Table 12-9 provides Performance index (PI) values of BPN′ singlevariants (constructed using PCR fusion as described in PCT App. No.PCT/US09/46156, filed Jun. 3, 2009, which is incorporated by referenceherein for such description) for stain removal in a BMI microswatchassay in Detergent Composition 2 at 16° C. and pH 8 and for stabilitymeasured in Detergent Composition 3. PI values for specific activity byAAPF hydrolysis (PI specific AAPF) were determined. All assays wereperformed as described in Example 1. Performance index values werecalculated relative to BPN wild type. PI values less than 0.01 areindicated as “0.01” in bold italics. “Det. Comp.” means DetergentComposition.

TABLE 12-9 Performance Index Values for BPN′ Single Variants BPN′ Det.Comp. 2 PI Specific Det. Comp. 3 Variant pH 8, 16° C., BMI PI AAPFStability PI S182E 1.34 1.05 0.50 N109I 1.28 1.22 0.20 N117H 1.15 0.250.20 K237D 1.15 0.60 0.70 L257Q 1.14 0.94 0.80 P225N 1.13 1.02 0.70S105H 1.11 1.07

S236I 1.10 0.58 0.90 L235H 1.10 0.65 0.70 S249E 1.07 0.72 0.30 N76E 1.070.76 0.20 S145N 1.06 1.16 1.10 N243D 1.05 1.03 0.90 R247N 1.04 1.04 0.50E195N 1.04 1.05 0.40 A98K 1.03 0.75 0.70 S182N 0.99 1.14 0.90 S161H 0.971.07 0.90 G83H 0.95 0.72 0.60 G131D 0.95 1.11 1.30 T71C 0.93 1.00 1.30K136Q 0.93 1.02 0.80 P40D 0.93 1.20 1.20 A187H 0.91 0.95 0.60 L250K 0.901.02 0.40 S9I 0.87 0.20

N76M 0.85 0.60 0.60 S132D 0.85 0.88 0.70 Q19F 0.83 0.47 0.30 E112H 0.831.02

S249P 0.80 1.02 0.50 S53D 0.78 0.29 0.10 V68E 0.78 0.59 1.70 D41I 0.721.15 0.90 K43H 0.70 0.28 0.10 V4H 0.66 0.37 0.60 A13Y 0.64 0.48

N62P 0.61 0.60 1.30 L196E 0.56 0.70 0.70 V44D 0.51 0.18

The invention includes a protease variant having proteolytic activity,the variant comprising an amino acid sequence having at least 60%, 70%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identityto SEQ ID NO:2 or SEQ ID NO:6 and at least one substitution selectedfrom those listed in Table 12-9, wherein positions of the variant arenumbered by correspondence with positions of the SEQ ID NO:2 sequence.Each such protease variant may be an isolated, recombinant,substantially pure, or non-naturally occurring protease variant. Alsoincluded are compositions, including cleaning compositions, comprisingat least one such protease variant and methods for cleaning utilizing atleast one such protease variant as described in greater detail elsewhereherein.

Example 13 Hand Dish Liquid Detergent Compositions

In this Example, various hand dish liquid detergent formulations areprovided. The following hand dish liquid detergent compositions of thepresent invention are provided below. In each of these formulations, atleast one protease variant provided herein is included at aconcentration of from about 0.0001 to about 10 weight percent. In somealternative aspects, other concentrations will find use, as determinedby the formulator, based on their needs.

TABLE 13-1 Light-Duty Liquid Dishwashing Detergent CompositionComposition 1 2 3 4 Linear Alkylbenzene Sulfonate (1) — — — Alkyl EthoxySulfate (2) 18%  17% 17%  18%  Paraffin Sulfonate (C15) — — — — CAP =coco amido propyl Betaine — — 9% 5% Nonionic (3) — — 1% — Amine Oxide(4)  6% 5.5% — 4% Alkylpolyglucoside 4% Alcohol (5) — — 5% 7% Pura =polypropyleneglycol  1% 0.8% — — Citrate — — 0.3%  0.6%  Salt (6) 1.2% 1.0% — 0.5%  SCS = sodium cumene sulfonate — — 0.8%  — glycerol 15%  5%3% — Na-lactate — — — 5% cationic polymer (7) 0.1%  0.1% 0.3%  0.2% Protease of this invention    0.0075    0.0050    0.0025    0.030 Glycoldistearate from Euperlan ® Cognis   0.4 0   0.4 0 Hydrogenated CastorOil Thixcin ® Elementis 0   0.1 0   0.1 Mica (BASF Mearlin superfine) 0  0.05 0   0.05 Minors* Balance to 100% with water  pH 9 9 6 6 OptionalMinors*: dyes, opacifier, perfumes, preservatives, hydrotropes,processing aids, and/or stabilizers. (1) Linear Alkylbenzene Sulfonate:LAS: C11.4 (2) Alkyl Ethoxy Sulfate: AExS: (3) Nonionic: AlkylEthoxylate(4) Di-methyl coco alkyl amine oxide (5) Alcohol: Ethanol (6) Salt: NaCl(7) cationically modified hydroxyethyl cellulose (Polyquaternium-10 -UCARE LR-400 ex Amerchol).

Example 14 Liquid & Granular Laundry Detergents

This Example provides various formulations for liquid laundrydetergents. The following liquid laundry detergent formulations of thepresent invention are provided below. In each of these formulations, atleast one protease variant provided herein is included at aconcentration of from about 0.0001 to about 10 weight percent. In somealternative aspects, other concentrations will find use, as determinedby the formulator, based on their needs.

Liquid laundry detergent compositions suitable for front-loadingautomatic washing machines.

Composition (wt % of composition) Ingredient 1 2 3 4 5 6 7 8Alkylbenzene sulfonic acid 7 11 4.5 1.2 1.5 12.5 5.2 4 Sodium C₁₂₋₁₄alkyl ethoxy 3 2.3 3.5 4.5 4.5 7 18 1.8 2 sulfate C₁₄₋₁₅ alkyl8-ethoxylate 5 8 2.5 2.6 4.5 4 3.7 2 C₁₂ alkyl dimethyl amine oxide — —0.2 — — — — — C₁₂₋₁₄ alkyl hydroxyethyl dimethyl — — — 0.5 — — — —ammonium chloride C₁₂₋₁₈ Fatty acid 2.6 4 4 2.6 2.8 11 2.6 1.5 Citricacid 2.6 3 1.5 2 2.5 3.5 2.6 2 Cold Water Protease* 0.05 0.03 0.04 0.030.04 0.03 0.03 0.02 Amylase (Natalase ®) 0.1 0.2 0.15 — 0.05 0.5 0.1 0.2Mannanase (Mannaway ®) 0.05 0.1 0.05 — — 0.1 0.04 — Random graftco-polymer¹ 1 0.2 1 0.4 0.5 2.7 0.3 1 A compound having the following0.4 2 0.4 0.6 1.5 1.8 0.7 0.3 general structure:bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)- bis((C₂H₅O)(C₂H₄O)n),wherein n = from 20 to 30, and x = from 3 to 8, or sulphated orsulphonated variants thereof Ethoxylated Polyethylenimine² — — — — — 0.5— — Amphiphilic alkoxylated grease 0.1 0.2 0.1 0.2 0.3 0.3 0.2 0.3cleaning polymer³ Diethoxylated poly (1,2 propylene — — — — — — 0.3 —terephthalate short block soil release polymer.Diethylenetriaminepenta(methylenephosphonic) 0.2 0.3 — — 0.2 — 0.2 0.3acid Hydroxyethane diphosphonic acid — — 0.45 — — 1.5 — 0.1 FWA 0.1 0.20.1 — — 0.2 0.05 0.1 Solvents (1,2 propanediol, 3 4 1.5 1.5 2 4.3 2 1.5ethanol), stabilizers Hydrogenated castor oil derivative 0.4 0.4 0.3 0.10.3 — 0.4 0.5 structurant Boric acid 1.5 2.5 1.5 1.5 0.5 1.5 1.5 Naformate — — — 1 — — — — Reversible protease inhibitor⁴ — — 0.002 — — — —— Perfume 0.5 0.7 0.5 0.5 0.8 1.5 0.5 0.8 Perfume MicroCapsules slurry0.2 0.3 0.7 0.2 0.05 0.4 0.9 0.7 (30% am) Ethoxylated thiophene Hueing0.005 0.007 0.010 0.008 0.008 0.007 0.007 0.008 Dye⁵ Buffers (sodiumhydroxide, To pH 8.2 Monoethanolamine) Water and minors (antifoam, To100% aesthetics)Liquid laundry detergent compositions suitable for top-loading automaticwashing machines.

Composition (wt % of composition) Ingredient 9 10 11 12 13 14 15 16C₁₂₋₁₅ 20.1 15.1 20.0 15.1 13.7 16.7 10.0 9.9 Alkylethoxy(1.8)sulfateC_(11.8) Alkylbenzene sulfonate 2.7 2.0 1.0 2.0 5.5 5.6 3.0 3.9 C₁₆₋₁₇Branched alkyl sulfate 6.5 4.9 4.9 3.0 9.0 2.0 C₁₂₋₁₄ Alkyl-9-ethoxylate0.8 0.8 0.8 0.8 8.0 1.5 0.3 11.5 C₁₂ dimethylamine oxide 0.9 Citric acid3.8 3.8 3.8 3.8 3.5 3.5 2.0 2.1 C₁₂₋₁₈ fatty acid 2.0 1.5 2.0 1.5 4.52.3 0.9 Cold Water Protease* 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.1 Amylase(Natalase ®) 0.7 0.3 0.6 0.3 0.6 0.4 Amylase (Termamyl Ultra ®) 1.1Mannanase (Mannaway ®) 0.1 0.1 Pectate Lyase (Pectawash ®) 0.1 0.2 Borax3.0 3.0 2.0 3.0 3.0 3.3 Na & Ca formate 0.2 0.2 0.2 0.2 0.7 A compoundhaving the 1.6 1.6 3.0 1.6 2.0 1.6 1.3 1.2 following general structure:bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)- bis((C₂H₅O)(C₂H₄O)n),wherein n = from 20 to 30, and x = from 3 to 8, or sulphated orsulphonated variants thereof Random graft co-polymer¹ 0.4 0.2 1.0 0.50.6 1.0 0.8 1.0 Diethylene triamine 0.4 0.4 0.4 0.4 0.2 0.3 0.8pentaacetic acid Tinopal AMS-GX 0.2 0.2 0.2 0.2 0.2 0.3 0.1 TinopalCBS-X 0.1 0.2 Amphiphilic alkoxylated 1.0 1.3 1.3 1.4 1.0 1.1 1.0 1.0grease cleaning polymer³ Texcare 240N (Clariant) 1.0 Ethanol 2.6 2.6 2.62.6 1.8 3.0 1.3 Propylene Glycol 4.6 4.6 4.6 4.6 3.0 4.0 2.5 Diethyleneglycol 3.0 3.0 3.0 3.0 3.0 2.7 3.6 Polyethylene glycol 0.2 0.2 0.2 0.20.1 0.3 0.1 1.4 Monoethanolamine 2.7 2.7 2.7 2.7 4.7 3.3 1.7 0.4Triethanolamine 0.9 NaOH to pH to pH to pH to pH to pH to pH to pH to pH8.3 8.3 8.3 8.3 8.3 8.3 8.3 8.5 Suds suppressor Dye 0.01 0.01 0.01 0.010.01 0.01 0.0 Perfume 0.5 0.5 0.5 0.5 0.7 0.7 0.8 0.6 PerfumeMicroCapsules 0.2 0.5 0.2 0.3 0.1 0.3 0.9 1.0 slurry (30% am)Ethoxylated thiophene 0.003 0.002 0.002 0.005 0.002 0.004 0.004 0.003Hueing Dye⁵ Water balance balance balance balance balance balancebalance balance

Granular detergent compositions produced in accordance with theinvention suitable for laundering fabrics.

17 18 19 20 21 22 Linear alkylbenzenesulfonate 15  12  20  10  12  13 with aliphatic carbon chain length C₁₁-C₁₂ Other surfactants 1.6 1.2 1.93.2 0.5 1.2 Phosphate builder(s) 2  3  4  Zeolite 1  1  4  1  Silicate4  5  2  3  3  5  Sodium Carbonate 2  5  5  4  0  3  Polyacrylate (MW4500) 1  0.6 1  1  1.5 1 Carboxymethyl cellulose 1  — 0.3 — 1.1 —(Finnfix BDA ex CPKelco) Celluclean ®  0.23  0.17 0.5 0.2 0.2 0.6 ColdWater Protease*  0.23  0.17 0.5 0.2 0.2 0.6 Stainzyme Plus ®  0.23  0.170.5 0.2 0.2 0.6 Fluorescent Brightener(s)  0.16  0.06  0.16  0.18  0.16 0.16 Diethylenetriamine pentaacetic 0.6 0.6  0.25 0.6 0.6 acid orEthylene diamine tetraacetic acid MgSO₄ 1  1  1  0.5 1 1 Bleach(es) andBleach  6.88  6.12  2.09  1.17  4.66 activator(s) Ethoxylated thiopheneHueing  0.002  0.001  0.003  0.003 — — Dye⁵ Direct Violet 9 ex CibaSpecialty   0.0006   0.0004   0.0006 Chemicals Sulfate/CitricAcid/Sodium Balance to 100% Bicarbonate/Moisture/perfume ¹Random graftcopolymer is a polyvinyl acetate grafted polyethylene oxide copolymerhaving a polyethylene oxide backbone and multiple polyvinyl acetate sidechains. The molecular weight of the polyethylene oxide backbone is about6000 and the weight ratio of the polyethylene oxide to polyvinyl acetateis about 40 to 60 and no than 1 grafting point per 50 ethylene oxideunits. ²Polyethylenimine (MW = 600) with 20 ethoxylate groups per —NH.³Amphiphilic alkoxylated grease cleaning polymer is a polyethylenimine(MW = 600) with 24 ethoxylate groups per —NH and 16 propoxylate groupsper —NH ⁴Reversible Protease inhibitor of structure:

⁵Ethoxylated thiophene Hueing Dye is a described in U.S. Pat. No.7,208,459 B2. *Remark: all enzyme levels expressed as % enzyme rawmaterial, except for cold water protease (of this invention) which isexpressed as % of active protein added to the product. .

Example 15 Unit Dose Compositions

This Example provides various formulations for unit dose laundrydetergents. Such unit dose formulations can comprise one or multiplecompartments.

The following unit dose laundry detergent formulations of the presentinvention are provided below.

TABLE 15-1 Unit Dose Compositions Ingredients 1 2 3 4 5 Alkylbenzenesulfonic acid C 11-13, 14.5 14.5 14.5 14.5 14.5 23.5% 2-phenyl isomerC₁₂₋₁₄ alkyl ethoxy 3 sulfate 7.5 7.5 7.5 7.5 7.5 C₁₂₋₁₄ alkyl7-ethoxylate 13.0 13.0 13.0 13.0 13.0 Citric Acid 0.6 0.6 0.6 0.6 0.6Fatty Acid 14.8 14.8 14.8 14.8 14.8 Enzymes (as % raw material not 1.71.7 1.7 1.7 1.7 active) Protease of this invention (as % 0.05 0.1 0.020.03 0.03 active) Ethoxylated Polyethylenimine¹ 4.0 4.0 4.0 4.0 4.0Series 1 GG36 protease (as % active) 0.02 0 0.01 0.02 0.03 Hydroxyethanediphosphonic acid 1.2 1.2 1.2 1.2 1.2 Brightener 0.3 0.3 0.3 0.3 0.3P-diol 15.8 13.8 13.8 13.8 13.8 Glycerol 6.1 6.1 6.1 6.1 6.1 MEA 8.0 8.08.0 8.0 8.0 TIPA — — 2.0 — — TEA — 2.0 — — — Cumene sulphonate — — — —2.0 cyclohexyl dimethanol — — — 2.0 — Water 10 10 10 10 10 Structurant0.14 0.14 0.14 0.14 0.14 Perfume 1.9 1.9 1.9 1.9 1.9 Buffers(monoethanolamine) To pH 8.0 Solvents (1,2 propanediol, ethanol) To 100%¹Polyethylenimine (MW = 600) with 20 ethoxylate groups per —NH.

Multiple compartment unit dose laundry detergent formulations of thepresent invention are provided below. In these examples the unit dosehas three compartments, but similar compositions can be made with two,four or five compartments. The film used to encapsulate the compartmentsis polyvinyl alcohol.

TABLE 15-2 Multiple Compartment Unit Dose Detergent Compositions BaseComposition 1 Ingredients % Glycerol (min 99) 5.3 1,2-propanediol 10Citric Acid 0.5 Monoethanolamine 10 Caustic soda — Dequest 2010 1.1Potassium sulfite 0.2 Nonionic Marlipal C24EO7 20.1 HLAS (surfactant)24.6 Optical brightener FWA49 0.2 C12-15 Fatty acid 16.4 PolymerLutensit Z96 2.9 Polyethyleneimine ethoxylate 1.1 PEI600 E20 MgCl2 0.2Solvents (1,2 propanediol, ethanol) To 100% Multi-compartmentformulations Composition 1 2 Compartment A B C A B C Volume 40 ml 5 ml 5ml 40 ml 5 ml 5 ml of each compartment Active material in Wt. % Perfume1.6 1.6 1.6 1.6 1.6 1.6 Dyes <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 TiO20.1 — — — 0.1 — Sodium Sulfite 0.4 0.4 0.4 0.3 0.3 0.3 Acusol 305, 1.2 2— — Rohm&Haas Hydrogenated 0.14 0.14 0.14 0.14 0.14 0.14 castor oil BaseAdd to Add to Add to Add to Add to Add to Composition 1 100% 100% 100%100% 100% 100%

Example 16 Cleaning Performance of BPN′-v36 Polypeptide Variants

BPN′-v36 polypeptide variants comprising two amino acid substitutionswere constructed by standard PCR fusion using the BPN′-v36 variant as abackbone or parent sequence. For this purpose, two or three partiallyoverlapping fragments were amplified by mutagenic primers prepared suchthat the primer encoded a desired substitution. PCR amplificationreactions were carried out as described in Example 7 of Part I supra.The following BPN′-v36 double mutant variants (i.e., BPN′-v36 with thefollowing two amino acid substitution) were constructed: Q019R-N025D,A001Y-Q275R, V004A-S249N, V004E-S260P, V004A-T55A, Y006F-S249C,Y006D-T55A, V008L-Q275R, Q010R-Q275K, L016Q-Q217H, H017R-T158A,S183D-Q206R, P210S-N212D, S018Y-V203A, S018K-V203I, Y021H-D259G,Y021H-D259R, K027R-N269D, K027R-N269T, S037P-S260F, S037T-S260P,D041E-N077D, D041G-N077E, G166V-S183T, N252S-L257H, V044A-Q206H,V044A-Q206K, V044A-Q206R, N076T-N212D, N076P-N212S, N077D-N252D,N077D-N252T, K141I-S248N, T158I-D259N, T158A-D259P, S161E-Q185H,K237M-H238R, G160A-D259G, G160R-D259V, G215R-D259R, G215D-D259V,N061D-Q206R, N061L-Q206H, S009L-N218S, S161E-S260T, Q019A-N109S,T022S-G166V, Y021H-N252H, P129S-K136R, T022S-T242S, N025K-H238R,N025D-Q185R, S037G-Q275H, K043R-N076S, K043N-Q217R, K043N-S163T,T055A-V147A, N061K-N252K, N062Y-G097D, Y021H-V084E, Y021H-S037E,N062Y-T244A, K027E-Y091F, A074S-P129Q, S249R-Q275R, I079V-Q217H,A098T-T158A, K027R-D120H, Q019R-Q185R, G131S-K265N, A133V-D259N,A144H-T244A, I035V-K043N, G160R-T244A, S161P-T253A, S163T-Q245L,K170R-D259G, S183T-S249R, N184Y-Y262N, V198L-D259G, A200T-H226L,Q206R-S260P, G211V-T244A, Q217R-T244A, L75I-N76D, S260P-Q275L,S260P-Q275R, Y262N-Q275R, V004A-Y006F, H017L-Q019A, N025D-V026A,N118G-V121A, V072F-L075I, S183T-R186K, V203A-Q217R, and S249R-Y262H. Thecleaning performance of these variants was tested in a BMI microswatchcleaning assay in Detergent Composition 4 at pH 8 and 16° C. and eggmicroswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.as described in Example 1 of Part I. Results are provided below.

The following BPN′ protease variants were determined to have a PI valueequal to or greater than 0.9 and equal or less than 1.0 relative toBPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acidsequence (SEQ ID NO:6) comprising at least one set of amino acidsubstitutions selected from the group consisting of BPN′-v3, BPN′-v36,S183T-S249R, N61D-Q206R, Y262N-Q275R, K43R-N76S, S183T-S249R,K170R-D259G, Y6F-S249C, Q19A-N109S, H17L-Q19A, Q19R-Q185R, S18Y-V203A,N61D-Q206R, S161E-S260T, S18K-V203I, V4A-T55A, N252S-L257H, S249R-Y262H,N61L-Q206H, N184Y-Y262N, Q19R-N25D, S249R-Y262H, A74S-P129Q, H17L-Q19A,K27R-D120H, V4A-T55A, Y21H-N252H, K27R-N269D, K27R-N269D, A98T-T158A,I79V-Q217H, S9L-N218S, V4A-Y6F, S161P-T253A, V203A-Q217R, T22S-T242S,N76P-N212S, K170R-D259G, S37T-S260P, T55A-V147A, Q19R-Q185R, V4A-Y6F,Q19A-N109S, Y262N-Q275R, G160R-T244A, Q19R-N25D, N25D-Q185R, A98T-T158A,N61L-Q206H, G211V-T244A, S9L-N218S, A144H-T244A, A144H-T244A,S18Y-V203A, Y21H-N252H, A74S-P129Q, A1Y-Q275R, V198L-D259G, T55A-V147A,K141I-S248N, S183T-R186K, S37T-S260P, K27R-D120H, T22S-T242S,S161E-Q185H, P129S-K136R, G211V-T244A, N76P-N212S, K43N-S163T,S37G-Q275H, S161P-T253A, Y6F-S249C, N184Y-Y262N, N252S-L257H,G160R-T244A, S37G-Q275H, P129S-K136R, N62Y-T244A, and S260P-Q275R,wherein amino acid positions of the variant are numbered bycorrespondence with the sequence of SEQ ID NO:2. Such variants haveenhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and agreater PI value than BPN′ in this assay. The invention includes aprotease variant having enhanced proteolytic activity compared to BPN′(SEQ ID NO:2), a PI value of equal to or greater than 0.9 and equal toor less than 1.0 relative to BPN′-v36 in this assay, the variantcomprising an amino acid sequence having at least 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ IDNO:2 or SEQ ID NO:6 and comprising at least one set of amino acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such protease variant as described in greater detail elsewhereherein.

Also provided is a subtilisin protease variant having enhancedproteolytic activity compared to BPN′ in a BMI microswatch cleaningassay in Detergent Composition 4 at pH 8 and 16° C., the variantcomprising an amino acid sequence having at least 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein thevariant comprises at least one substitution comprising at least onesubstitution selected from the group of X1Y, X4A, X6F, X9L, X17L,X18K/Y, X19A/R, X21H, X22S, X25D, X27R, X37G/T, X43N/R, X55A, X61D/L,X62Y, X74S, X76P/S, X79V, X98T, X109S, X120H, X129Q/S, X136R, X141I,X144H, X147A, X158A, X160R, X161E/P, X163T, X170R, X183T, X184Y,X185H/R, X186K, X198L, X203A/I, X206H/R, X211V, X212S, X217H/R, X218S,X242S, X244A, X248N, X249C/R, X252H/S, X253A, X257H, X259G, X260P/T,X262H/N, X269D, and X275H/R, and optionally at least one substitutionselected from the group of A1Y, V4A, Y6F, S9L, H17L, S18K/Y, Q19A/R,Y21H, T22S, N25D, K27R, S37G/T, K43N/R, T55A, N61D/L, N62Y, A74S,N76P/S, I79V, A98T, N109S, D120H, P129Q/S, K136R, K141I, A144H, V147A,T158A, G160R, S161E/P, S163T, K170R, S183T, N184Y, Q185H/R, R186K,V198L, V203A/I, Q206H/R, G211V, N212S, Q217H/R, N218S, T242S, T244A,S248N, S249C/R, N252H/S, T253A, L257H, D259G, S260P/T, Y262H/N, N269D,and Q275H/R, wherein amino acid positions of the variant are numbered bycorrespondence with positions of the sequence of SEQ ID NO:2. Suchvariants have enhanced proteolytic activity compared to the BPN′ (SEQ IDNO:2) BPN′-v3, and BPN′-v36 and a PI value greater than that of BPN′,BPN′-v3, and BPN′-v36 in this assay. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such protease variant as described in greater detail elsewhereherein.

The following BPN′ subtilisin protease variants were determined to havea PI value equal or greater than 0.5 and less than 0.9 relative toBPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acidsequence (SEQ ID NO:6) comprising at least one set of amino acidsubstitutions selected from the group consisting of Y21H-D259G,Y21H-D259G, A133V-D259N, I79V-Q217H, S18K-V203I, T158A-D259P,N61K-N252K, K43N-Q217R, T158A-D259P, Q206R-S260P, A133V-D259N,V198L-D259G, N61K-N252K, S161E-S260T, G160A-D259G, K43N-Q217R,A1Y-Q275R, A200T-H226L, Q217R-T244A, S260P-Q275R, Q206R-S260P,T158I-D259N, Q217R-T244A, L75I-N76D, S161E-Q185H, Y21H-S37E,S249R-Q275R, G160A-D259G, T158I-D259N, Y21H-S37E, N76T-N212D,S260P-Q275L, G131S-K265N, V4A-S249N, N25D-Q185R, K43R-N76S, S183D-Q206R,Q10R-Q275K, K43N-S163T, Q10R-Q275K, N25D-V26A, G131S-K265N, S260P-Q275L,K141I-S248N, L16Q-Q217H, S249R-Q275R, K27R-N269T, P210S-N212D,L75I-N76D, S183D-Q206R, N118G-V121A, G215D-D259V, N76T-N212D, V4A-S249N,K27R-N269T, N62Y-G97D, V4E-S260P, G215D-D259V, K27E-Y91F, Y6D-T55A,N77D-N252T, V4E-S260P, Y6D-T55A, N77D-N252T, N25K-H238R, V44A-Q206H,L16Q-Q217H, S37P-S260F, V44A-Q206R, V44A-Q206H, V44A-Q206R, andS37P-S260F, wherein amino acid positions of the variant are numbered bycorrespondence with the sequence of SEQ ID NO:2. Such variants haveproteolytic activity. The invention includes a protease variant havingproteolytic activity, having enhanced proteolytic activity greater thanBPN′, and/or a PI value equal to or greater than 0.5 and less than 0.9relative to BPN′-v36 in this assay, the variant comprising an amino acidsequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 andcomprising at least one set of acid substitutions selected from saidgroup above, wherein amino acid positions of the variant are numbered bycorrespondence with amino acid positions of the SEQ ID NO:2 sequence.Also included are compositions, including, but not limited to, e.g.,cleaning compositions, comprising at least one such variant and methodsfor cleaning utilizing at least one such protease variant as describedin greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI valuegreater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from greater than 1.0 to about 10, from greater than1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence(SEQ ID NO:6) comprising at least one set of amino acid substitutionsselected from the group consisting of Y21H-D259G, S183T-S249R,N61D-Q206R, Y262N-Q275R, Y021H-D259G, K043R-N076S, S183T-S249R,A133V-D259N, and I079V-Q217H, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have enhanced proteolytic activity compared to the BPN′,BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3 andBPN′-v36 in this assay. The invention includes a protease variant havingenhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), enhancedproteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, a PI valueof greater than 1.0 to about 5 relative to BPN′-v3, and/or a PI value ofgreater than 1.0 to about 5 relative to BPN′-v36 in this assay, thevariant comprising an amino acid sequence having at least 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity toSEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such protease variant as described in greater detail elsewhereherein.

Also provided is a subtilisin protease variant having enhancedproteolytic activity compared to BPN′ in a BMI microswatch cleaningassay in Detergent Composition 4 at pH 8 and 16° C., the variantcomprising an amino acid sequence having at least 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein thevariant comprises at least one substitution comprising at least onesubstitution selected from the group of X021H, X043R, X061D, X076S,X079V, X133V, X183T, X206R, X217H, X249R, X259G/N, X262N, and X275R, andoptionally at least one substitution selected from the group of Y021H,K043R, N061D, N076S, I079V, A133V, S183T, Q206R, Q217H, S249R, D259G/N,Y262N, and Q275R, wherein amino acid positions of the variant arenumbered by correspondence with positions of the sequence of SEQ IDNO:2. Such variants have enhanced proteolytic activity compared to theBPN′ (SEQ ID NO:2) BPN′-v3, and BPN′-v36 and a PI value greater thanthat of BPN′, BPN′-v3, and BPN′-v36 in this assay. Also included arecompositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such protease variant as described ingreater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equalto or greater than 0.5 and less than or equal to 1.0 relative toBPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acidsequence (SEQ ID NO:6) comprising at least one set of amino acidsubstitutions selected from the group consisting of BPN′-v3, BPN′-v36,K170R-D259G, S18K-V203I, Y6F-S249C, Q19A-N109S, H17L-Q19A, Q19R-Q185R,S18Y-V203A, N61D-Q206R, S161E-S260T, S18K-V203I, V4A-T55A, N252S-L257H,S249R-Y262H, N61L-Q206H, N184Y-Y262N, Q19R-N25D, S249R-Y262H,A74S-P129Q, T158A-D259P, H17L-Q19A, K27R-D120H, V4A-T55A, N61K-N252K,Y21H-N252H, K27R-N269D, K43N-Q217R, T158A-D259P, Q206R-S260P,K27R-N269D, A98T-T158A, I79V-Q217H, S9L-N218S, V4A-Y6F, S161P-T253A,V203A-Q217R, T22S-T242S, N76P-N212S, A133V-D259N, K170R-D259G,S37T-S260P, T55A-V147A, V198L-D259G, Q19R-Q185R, V4A-Y6F, Q19A-N109S,Y262N-Q275R, G160R-T244A, Q19R-N25D, N25D-Q185R, N61K-N252K,S161E-S260T, A98T-T158A, N61L-Q206H, G211V-T244A, S9L-N218S,A144H-T244A, A144H-T244A, S18Y-V203A, Y21H-N252H, A74S-P129Q,G160A-D259G, K43N-Q217R, A1Y-Q275R, A1Y-Q275R, A200T-H226L, Q217R-T244A,S260P-Q275R, V198L-D259G, T55A-V147A, Q206R-S260P, K141I-S248N,S183T-R186K, T158I-D259N, S37T-S260P, K27R-D120H, T22S-T242S,Q217R-T244A, S161E-Q185H, P129S-K136R, G211V-T244A, N76P-N212S,L75I-N76D, S161E-Q185H, Y21H-S37E, S249R-Q275R, G160A-D259G, K43N-S163T,T158I-D259N, Y21H-S37E, S37G-Q275H, S161P-T253A, N76T-N212D,S260P-Q275L, Y6F-S249C, N184Y-Y262N, G131S-K265N, V4A-S249N, N25D-Q185R,N252S-L257H, K43R-N76S, S183D-Q206R, G160R-T244A, Q10R-Q275K,S37G-Q275H, K43N-S163T, Q10R-Q275K, N25D-V26A, P129S-K136R, G131S-K265N,S260P-Q275L, K141I-S248N, T22S-G166V, N62Y-T244A, L16Q-Q217H,S249R-Q275R, S260P-Q275R, K27R-N269T, P210S-N212D, L75I-N76D,S183D-Q206R, N118G-V121A, G215D-D259V, N76T-N212D, V4A-S249N,K27R-N269T, G166V-S183T, N62Y-G97D, V4E-S260P, G215D-D259V, K27E-Y91F,Y21H-D259R, Y6D-T55A, N77D-N252T, V4E-S260P, Y6D-T55A, N77D-N252T,Y21H-D259R, N25K-H238R, N77D-N252D, V44A-Q206H, L16Q-Q217H, V72F-L75I,S37P-S260F, V72F-L75I, N77D-N252D, V44A-Q206R, S163T-Q245L, V44A-Q206H,V44A-Q206R, S37P-S260F, G215R-D259R, V44A-Q206K, and V44A-Q206K, whereinamino acid positions of the variant are numbered by correspondence withthe sequence of SEQ ID NO:2. Such variants have proteolytic activity.The invention includes a protease variant having proteolytic activityand/or a PI value equal to or greater than 0.5 and equal to or less than1.0 relative to BPN′-v36 in this assay, the variant comprising an aminoacid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6and comprising at least one set of acid substitutions selected from saidgroup above, wherein amino acid positions of the variant are numbered bycorrespondence with amino acid positions of the SEQ ID NO:2 sequence.Also included are compositions, including, but not limited to, e.g.,cleaning compositions, comprising at least one such variant and methodsfor cleaning utilizing at least one such protease variant as describedin greater detail elsewhere herein.

Example 17 Cleaning Performance of Additional BPN′-v36 PolypeptideVariants

The following BPN′-v36 variants were synthesized at DNA2.0 (Menlo Park,Calif.) using the pHPLT-BPN′-v36 plasmid containing the BPN′ expressioncassette served as template DNA (parent plasmid) for cloning:N109G-A128S-S224A, N109G-A128S-S224A-N243V,A88T-N109G-A116T-A128S-S224A-N243V, N61G-N109G-A128S-S224A,N61G-N109G-A128S-S224A-N243V, N61G-A88T-N109G-A116T-A128S-S224A-N243V,N109Q-A128S-S224A,

N109Q-A128S-S224A-N243V, A88T-N109Q-A116T-A128S-S224A-N243V,N109S-A128S-S224A, N109S-A128S-S224A-N243V,A88T-N109S-A116T-A128S-S224A-N243V, N109M-A128S-S224A,N109M-A128S-S224A-N243V, A88T-N109M-A116T-A128S-S224A-N243V,N109G-A114S-A128S, N109G-A114S-A128S-N243V,A88T-N109G-A114S-A116T-A128S-N243V, N109G-A114S-A128S-S224A,N109G-A114S-A128S-S224A-N243V, N109G-A128S-S183V, N109G-A128S-S183L,N109G-A128S-S183L-S224A, N109G-A114S-A128S-S183L-S224A,A88T-N109G-A114S-A116T-A128S-S183L-S224A-N243V, N76D-N109G-A128S-S224A,N101Q-N109Q-A128S-S224A-N243V,N101Q-N109Q-A128S-P129S-S130T-S224A-N243V,N109G-A128S-P129S-S130T-S224A-N243V, S33T-A128S-N218S,S33T-N109G-A128S-N218S-N243V, S33T-N61G-N109G-A128S-N218S-N243V,S33T-N109G-A128S-G169A-N218S-N243V, S33T-S63G-N109G-A128S-N218S-N243V,S33T-N76D-N109G-A128S-N218S-N243V,S33T-S63G-N109G-A128S-G169A-N218S-N243V,I31L-S33T-S63G-N109G-A128S-G169A-N218S-N243V,S33T-N61G-S63G-N109G-A128S-N218S-N243V,S33T-N61G-A88T-N109G-A116T-A128S-N218S-N243V,S33T-N61G-S63G-N109G-A128S-G131H-G169A-N218S-N243V,S33T-N61P-S63G-N109G-A128S-G131H-G169A-N218S-N243V,S33T-N109G-A128S-N218S-S224A-N243V, S63G-N109Q-A128S-S224A-N243V,S63G-N109Q-A128S-G131H-S224A-N243V, N61P-S63G-N109Q-A128S-S224A-N243V,N61P-S63G-N109Q-A128S-G131H-S224A-N243V,A1G-N61P-S63G-N109Q-A128S-G131H-S224A-N243V,I31L-N61P-S63G-N109Q-A128S-G131H-S224A-N243V,N61P-S63G-N109Q-A128S-G131H-S224A-N243V-S249Q,S33T-T55P-N61P-S63G-N109Q-A128S-G131H-S224A-N243V,S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V,S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V-S249Q,A1G-I31L-S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V-S249Q,N61P-S63G-N109Q-A128S-G131H-G169A-S224A-N243V-S249Q, andA1G-I31L-S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-G169A-S224A-N243V-S249Q.

The variants were grown for protein expression as described in Example11 of Part I. These variants were tested for their performance in theBMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and16° C., the egg microswatch cleaning assay in Detergent Composition 4 atpH 8 and 16° C., and the AAPF assay as described in Example 1 of Part I.Results are provided below.

The following BPN′-v36 variants were determined to have a PI valuegreater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from greater than 1.0 to about 10, from greater than1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence(SEQ ID NO:6) comprising at least one set of amino acid substitutionsselected from the group consisting of N61P-S63G-N109Q-A128S-S224A-N243V,A88T-N109G-A114S-A116T-A128S-N243V,A88T-N109G-A114S-A116T-A128S-S183L-S224A-N243V, N109G-A128S-S183V,N109G-A128S-N243V-K256R, N109M-A128S-S224A,A88T-N109S-A116T-A128S-S224A-N243V, N109Q-A128S-S224A-N243V,A88T-N109M-A116T-A128S-S224A-N243V, N109S-A128S-S224A-N243V,A88T-N109G-A116T-N243V, N101Q-N109Q-A128S-S224A-N243V,N109G-A116T-N243V-K256R, N109G-A128S-P129S-S130T-S224A-N243V, andA88T-N109Q-A116T-A128S-S224A-N243V, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have enhanced proteolytic activity compared to the BPN′,BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3 andBPN′-v36 in this assay. The invention includes a protease variant havingenhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), enhancedproteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, a PI valueof greater than 1.0 to about 5 relative to BPN′-v3, and/or a PI value ofgreater than 1.0 to about 5 relative to BPN′-v36 in this assay, thevariant comprising an amino acid sequence having at least 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity toSEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such protease variant as described in greater detail elsewhereherein.

Also provided is a subtilisin protease variant having enhancedproteolytic activity compared to BPN′-v36 and/or a PI value of equal toor greater than 1.0 compared to BPN′-v36 in a BMI microswatch cleaningassay in Detergent Composition 4 at pH 8 and 16° C., the variantcomprising an amino acid sequence having at least 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein thevariant comprises at least one substitution comprising at least onesubstitution selected from the group of X61G/P/S, X63G, X88T, X101Q,X109G/M/Q/S, X114S, X116T, X128S, X129S, X130T, X158S, X183L/V, X224A,X243V, X248A, and X256R, and optionally at least one substitutionselected from the group of N61G/P/S, S63G, A88T, N101Q, N109G/M/Q/S,A114S, A116T, A128S, P129S, S130T, T158S, S183L/V, S224A, N243V, S248A,and K256R, wherein amino acid positions of the variant are numbered bycorrespondence with positions of the sequence of SEQ ID NO:2. Suchvariants have enhanced proteolytic activity compared to the BPN′ (SEQ IDNO:2) BPN′-v3, and BPN′-v36 and a PI value greater than that of BPN′,BPN′-v3, and BPN′-v36 in this assay. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such protease variant as described in greater detail elsewhereherein.

The following BPN′-v36 variants were determined to have a PI value equalto or greater than 0.9 and equal to or less than 1.0 relative toBPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acidsequence (SEQ ID NO:6) comprising at least one set of amino acidsubstitutions selected from the group consisting ofG24S-G53S-N78S-G97A-N101S-A128S, G24S-G53S-N78S-G97A-N101S, BPN′-v36,S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V,S33T-N61G-S63G-N109G-A128S-N218S-N243V,S33T-S63G-N109G-A128S-N218S-N243V,S33T-T55P-N61P-S63G-N109Q-A128S-G131H-S224A-N243V,N61P-S63G-N109Q-A128S-G131H-G169A-S224A-N243V-S249Q,S33T-N61G-A88T-N109G-A116T-A128S-N218S-N243V,S33T-N109G-A128S-N218S-N243V, S33T-N76D-N109G-A128S-N218S-N243V,S33T-N76D-N109G-A128S-N218S-N243V-S248N-K256R,S33T-N61G-N109G-A128S-N218S-N243V, S33T-A128S-N218S,A1G-N61P-S63G-N109Q-A128S-G131H-S224A-N243V,N61P-S63G-N109Q-A128S-G131H-S224A-N243V-S249Q,N61P-S63G-N109Q-A128S-G131H-S224A-N243V,S63G-N109Q-A128S-G131H-S224A-N243V, N109G-A114S-A128S,N109G-A114S-A128S-S183L-S224A, N109G-A114S-A128S-S224A,N109G-A114S-A128S-S224A-N243V, A88T-N109G-A116T-A128S-S224A-N243V,N61G-A88T-N109G-A116T-A128S-S224A-N243V, N109G-A114S-A128S-N243V,N109G-A128S-S224A-N243V, N109G-A128S-S224A, N109G-A128S-S183L-S224A,N61G-N109G-A128S-S224A, N109G-A128S-S183L, S33T-N76D, N109S-A128S-S224A,N101Q-N109Q-A128S-P129S-S130T-S224A-N243V, S63G-N109Q-A128S-S224A-N243V,N109M-A128S-S224A-N243V, S63G-N109G, N109G-K256R, S63G-N76D,S33T-N109G-A128S-G169A-N218S-N243V, andS33T-N109G-A128S-N218S-S224A-N243V, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have proteolytic activity. The invention includes aprotease variant having proteolytic activity, enhanced proteolyticactivity compared to BPN′, and/or a PI value equal to or greater than0.9 and less or equal to 1.0 relative to BPN′-v36 in this assay, thevariant comprising an amino acid sequence having at least 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity toSEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such protease variant as described in greater detail elsewhereherein.

The following BPN′-v36 variants were determined to have a PI value equalto or greater than 0.5 and less than 0.9 relative to BPN′-v36 in a BMImicroswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of131L-S33T-S63G-N109G-A128S-G169A-N218S-N243V,A1G-I31L-S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-G169A-S224A-N243V-S249Q,S33T-N61G-S63G-N109G-A128S-G131H-G169A-N218S-N243V,S33T-S63G-N109G-A128S-G169A-N218S-N243V,S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V-S249Q,S33T-N76D-A128S-N218S, N76D-N109G-A128S-S224A, andS33T-N61P-S63G-N109G-A128S-G131H-G169A-N218S-N243V, wherein amino acidpositions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. Such variants have proteolytic activity. Theinvention includes a protease variant having proteolytic activity and/ora PI value equal to or greater than 0.5 and less than 0.9 relative toBPN′-v36 in this assay, the variant comprising an amino acid sequencehaving at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprisingat least one set of acid substitutions selected from said group above,wherein amino acid positions of the variant are numbered bycorrespondence with amino acid positions of the SEQ ID NO:2 sequence.Also included are compositions, including, but not limited to, e.g.,cleaning compositions, comprising at least one such variant and methodsfor cleaning utilizing at least one such protease variant as describedin greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI valuegreater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from greater than 1.0 to about 10, from greater than1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence(SEQ ID NO:6) comprising at least one set of amino acid substitutionsselected from the group consisting ofS33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V,S33T-N61G-S63G-N109G-A128S-N218S-N243V,S33T-S63G-N109G-A128S-N218S-N243V,N61P-S63G-N109Q-A128S-G131H-G169A-S224A-N243V-S249Q,S33T-N61G-A88T-N109G-A116T-A128S-N218S-N243V,S33T-N109G-A128S-N218S-N243V, S33T-A128S-N218S,A1G-N61P-S63G-N109Q-A128S-G131H-S224A-N243V,N61P-S63G-N109Q-A128S-G131H-S224A-N243V-S249Q,S63G-N109Q-A128S-G131H-S224A-N243V, N61P-S63G-N109Q-A128S-S224A-N243V,A88T-N109G-A114S-A116T-A128S-N243V,A88T-N109G-A114S-A116T-A128S-S183L-S224A-N243V, N109G-A114S-A128S,N109G-A114S-A128S-S183L-S224A, N109G-A114S-A128S-S224A,N109G-A114S-A128S-S224A-N243V, A88T-N109G-A116T-A128S-S224A-N243V,N61G-A88T-N109G-A116T-A128S-S224A-N243V, N109G-A128S-S183V,N109G-A114S-A128S-N243V, N109G-A128S-N243V-S248A,N109G-A128S-S224A-N243V, N109G-A128S-N243V-K256R, N109G-A128S-S224A,N109G-A128S-S183L-S224A, N61G-N109G-A128S-S224A, N109M-A128S-S224A,A88T-N109S-A116T-A128S-S224A-N243V, N109M-A128S-S224A-N243V, S63G-A128S,A88T-N109G-A116T-N243V, N101Q-N109Q-A128S-S224A-N243V,N109G-A116T-N243V-K256R, N109G-A116T, S63G-N109G, A88T-N109G,N109G-K256R, N61G-N109G-N243V, S33T-N109G-A128S-G169A-N218S-N243V,S33T-N109G-A128S-N218S-S224A-N243V, N109G-A128S-P129S-S130T-S224A-N243V,and A88T-N109Q-A116T-A128S-S224A-N243V, wherein amino acid positions ofthe variant are numbered by correspondence with the sequence of SEQ IDNO:2. Such variants have enhanced proteolytic activity compared to theBPN′, BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3and BPN′-v36 in this assay. The invention includes a protease varianthaving enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2),enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, aPI value of greater than 1.0 to about 5 relative to BPN′-v3, and/or a PIvalue of greater than 1.0 to about 5 relative to BPN′-v36 in this assay,the variant comprising an amino acid sequence having at least 60%, 70%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identityto SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of aminoacid substitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such protease variant as described in greater detail elsewhereherein.

Also provided is a subtilisin protease variant having enhancedproteolytic activity compared to BPN′-v36 and/or a PI value of greaterthan 1.0 compared to BPN′-v36 in an egg microswatch cleaning assay inDetergent Composition 4 at pH 8 and 16° C., the variant comprising anamino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprisesat least one substitution comprising at least one substitution selectedfrom the group of X1G, X33T, X55P, X61G/P/S, X63G, X76D, X88T, X101Q,X109G/M/Q/S, X114S, X116T, X128S, X129S, X130T, X131H, X158S, X169A,X183L/V, X218S, X224A, X243V, X248A/N, X249Q, X256R, and optionally atleast one substitution selected from the group of A1G, S33T, T55P,N61G/P/S, S63G, N76D, A88T, N101Q, N109G/M/Q/S, A114S, A116T, A128S,P129S, S130T, G131H, T158S, G169A, S183L/V, N218S, S224A, N243V,S248A/N, S249Q, K256R, wherein amino acid positions of the variant arenumbered by correspondence with positions of the sequence of SEQ IDNO:2. Such variants have enhanced proteolytic activity compared to theBPN′ (SEQ ID NO:2) BPN′-v3, and BPN′-v36 and a PI value greater thanthat of BPN′, BPN′-v3, and BPN′-v36 in this assay. Also included arecompositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such protease variant as described ingreater detail elsewhere herein.

The following BPN′-v36 variant was determined to have a PI value equalto or greater than 0.5 and equal to or less than 1.0 relative toBPN′-v36 in an egg BMI microswatch cleaning assay in DetergentComposition 4 at pH 8 and 16° C.:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising amino acid substitutions selected from the groupconsisting of substitutions S33T-N76D-A128S-N218S,N76D-N109G-A128S-S224A, and S063G-N76D, wherein amino acid positions ofthe variant are numbered by correspondence with the sequence of SEQ IDNO:2. Such variants have proteolytic activity. The invention includes aprotease variant having proteolytic activity and/or a PI value equal toor greater than 0.5 and equal to or less than 1.0 relative to BPN′-v36in this assay, the variant comprising an amino acid sequence having atleast 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising amino acidsubstitutions selected from the group consisting ofS33T-N76D-A128S-N218S, N76D-N109G-A128S-S224A, and S063G-N076D, whereinamino acid positions of the variant are numbered by correspondence withamino acid positions of the SEQ ID NO:2 sequence. Also included arecompositions, including, but not limited to, e.g., cleaningcompositions, comprising at least one such variant and methods forcleaning utilizing at least one such protease variant as described ingreater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI valuegreater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, at least 2, from greater than 1.0 to about 10, from greater than1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36in an AAPF proteolytic assay: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Qamino acid sequence (SEQ ID NO:6) comprising at least one set of aminoacid substitutions selected from the group consisting ofG24S-G53S-N78S-G97A-N101S-A128S,I31L-S33T-S63G-N109G-A128S-G169A-N218S-N243V,A1G-I31L-S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-G169A-S224A-N243V-S249Q,S33T-N61G-S63G-N109G-A128S-G131H-G169A-N218S-N243V,S33T-S63G-N109G-A128S-G169A-N218S-N243V,S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V,S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V-S249Q,S33T-N61G-S63G-N109G-A128S-N218S-N243V,S33T-S63G-N109G-A128S-N218S-N243V,S33T-T55P-N61P-S63G-N109Q-A128S-G131H-S224A-N243V,N61P-S63G-N109Q-A128S-G131H-G169A-S224A-N243V-S249Q,S33T-N61G-A88T-N109G-A116T-A128S-N218S-N243V,S33T-N109G-A128S-N218S-N243V, S33T-N76D-N109G-A128S-N218S-N243V,S33T-N76D-N109G-A128S-N218S-N243V-S248N-K256R,S33T-N61G-N109G-A128S-N218S-N243V, S33T-N76D-A128S-N218S,S33T-A128S-N218S, A1G-N61P-S63G-N109Q-A128S-G131H-S224A-N243V,N61P-S63G-N109Q-A128S-G131H-S224A-N243V-S249Q,N61P-S63G-N109Q-A128S-G131H-S224A-N243V,S63G-N109Q-A128S-G131H-S224A-N243V, N61P-S63G-N109Q-A128S-S224A-N243V,A88T-N109G-A114S-A116T-A128S-N243V,A88T-N109G-A114S-A116T-A128S-S183L-S224A-N243V, N109G-A114S-A128S,N109G-A114S-A128S-S183L-S224A, N109G-A114S-A128S-S224A,N109G-A114S-A128S-S224A-N243V, A88T-N109G-A116T-A128S-S224A-N243V,N61G-A88T-N109G-A116T-A128S-S224A-N243V, N109G-A128S-S183V,N109G-A114S-A128S-N243V, N109G-A128S-N243V-S248A,N109G-A128S-S224A-N243V, N109G-A128S-N243V-K256R, N109G-A128S-S224A,N109G-A128S-S183L-S224A, N61G-N109G-A128S-S224A, N76D-N109G-A128S-S224A,N109M-A128S-S224A, N109G-A128S-S183L, S33T-N76D,A88T-N109S-A116T-A128S-S224A-N243V, N109Q-A128S-S224A-N243V,N109S-A128S-S224A, A88T-N109M-A116T-A128S-S224A-N243V,N101Q-N109Q-A128S-P129S-S130T-S224A-N243V, S63G-N109Q-A128S-S224A-N243V,N109M-A128S-S224A-N243V, S63G-A128S, N109S-A128S-S224A-N243V,A88T-N109G-A116T-N243V, N61S-N109G-N243V, N101Q-N109Q-A128S-S224A-N243V,N109G-A116T-N243V-K256R, A88T-N109G-A116T-T158S-N243V-K256R,N109G-A116T, S63G-N109G, A88T-N109G, N109G-K256R, N61G-N109G-N243V,S33T-N61P-S63G-N109G-A128S-G131H-G169A-N218S-N243V,S33T-N109G-A128S-G169A-N218S-N243V, S33T-N109G-A128S-N218S-S224A-N243V,N109G-A128S-P129S-S130T-S224A-N243V, andA88T-N109Q-A116T-A128S-S224A-N243V, wherein amino acid positions of thevariant are numbered by correspondence with the sequence of SEQ ID NO:2.Such variants have enhanced proteolytic activity compared to the BPN′,BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3 andBPN′-v36 in this assay. The invention includes a protease variant havingenhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), enhancedproteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, a PI valueof greater than 1.0 to about 5 relative to BPN′-v3, and/or a PI value ofgreater than 1.0 to about 5 relative to BPN′-v36 in this assay, thevariant comprising an amino acid sequence having at least 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity toSEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such protease variant as described in greater detail elsewhereherein.

Also provided is a subtilisin protease variant having enhancedproteolytic activity compared to BPN′-v36 and/or a PI value of greaterthan 1.0 compared to BPN′-v36 in An AAPF proteolytic assay, the variantcomprising an amino acid sequence having at least 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein thevariant comprises at least one substitution comprising at least onesubstitution selected from the group of X1G, X31L, X33T, X55P, X61G/P/S,X63G, X76D, X88T, X101Q, X109G/M/Q/S, X114S, X116T, X128S, X129S, X130T,X131H, X158S, X169A, X183L/V, X218S, X224A, X243V, X248A/N, X249Q, andX256R, and optionally at least one substitution selected from the groupof A1G, I31L, S33T, T55P, N61G/P/S, S63G, N76D, A88T, N101Q,N109G/M/Q/S, A114S, A116T, A128S, P129S, S130T, G131H, T158S, G169A,S183L/V, N218S, S224A, N243V, S248A/N, S249Q, and K256R, wherein aminoacid positions of the variant are numbered by correspondence withpositions of the sequence of SEQ ID NO:2. Such variants have enhancedproteolytic activity compared to the BPN′ (SEQ ID NO:2) BPN′-v3, andBPN′-v36 and a PI value greater than that of BPN′, BPN′-v3, and BPN′-v36in this assay. Also included are compositions, including, but notlimited to, e.g., cleaning compositions, comprising at least one suchvariant and methods for cleaning utilizing at least one such proteasevariant as described in greater detail elsewhere herein.

The following BPN′-v36 variant was determined to have a PI value equalto or greater than 0.5 and equal to or less than 1.0 relative toBPN′-v36 in an AAPF proteolytic assay:BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising amino acid substitutions G24S-G53S-N78S-G97A-N101S orS063G-N76D, wherein amino acid positions of the variant are numbered bycorrespondence with the sequence of SEQ ID NO:2. Such variants haveproteolytic activity. The invention includes a protease variant havingproteolytic activity and/or a PI value equal to or greater than 0.5 andequal to or less than 1.0 relative to BPN′-v36 in this assay, thevariant comprising an amino acid sequence having at least 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity toSEQ ID NO:2 or SEQ ID NO:6 and comprising substitutionsG24S-G53S-N78S-G97A-N101S or S063G-N76D, wherein amino acid positions ofthe variant are numbered by correspondence with amino acid positions ofthe SEQ ID NO:2 sequence. Also included are compositions, including, butnot limited to, e.g., cleaning compositions, comprising at least onesuch variant and methods for cleaning utilizing at least one suchprotease variant as described in greater detail elsewhere herein.

Example 18 Cleaning Performance of Polypeptide BPN′-v36 PolypeptidesVariants from Combinatorial Libraries Based on BPN′-v36 Polypeptide

Two separate combinatorial libraries (AJ1 and AJ2) were synthesized byDNA2.0 (Menlo Park, Calif.) and were delivered as individual ligationreactions. The pHPLT-BPN′-v36 plasmid (FIG. 4) containing the BPN′expression cassette served as template DNA (parent plasmid) for libraryconstruction. A list of the possible amino acid positions andsubstitutions for each library is shown in Table 23-1. The ligationreactions for each library were used to transform B. subtilis, and thelibrary variants were grown up for protein expression as described inExample 11 The variants were tested for performance in the BMImicroswatch cleaning assay in Detergent Composition 4 (from Table 1-3)at pH 8 and 16° C. as described in Example 1.

TABLE 18-1 Possible Substitutions for Combinatorial Libraries AJ1 andAJ2 AJ1 AJ2 Possible Possible Position Substitutions PositionSubstitutions S33 G, S E54 E, Q D60 D, G D99 D, N N62 N, L, S D120 D, NS63 S, R, L, N, G D140 D, N S125 S, A E156 E, Q Q217 Q, R, E, L, G D197D, N M222 M, L, S K12 K, T K27 K, S K43 K, T K141 K, Y K213 K, Q K237 K,A K256 K, Q

The following BPN′-v36 variants were determined to have a PI value equalto or greater than 0.5 and less or equal to 1.0 relative to BPN′-v36 inthe BMI microswatch cleaning assay in Detergent Composition 4 at pH 8and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence(SEQ ID NO:6) comprising at least one set of amino acid substitutionsselected from the group consisting of BPN′v36, BPN′v3(G024S-G053S-N078S-G097A-N101S), BPN′v3 (G024S-G053S-N078S-G097A-N101S,BPN′ v12 (G024S-G053S-N078S-G097A-N101S-A128S), N062L, N062L-S063G,N062S, N062S-S063G-Q217L, N062S-S063L-Q217L, N062S-S063N, N062S-S063R,Q217E, S063G, S063G-Q217L, S063G-Q217L-M222S, S063L-Q217L, S063N,S063N-Q217L, D099N-K141Y-K213Q, D099N-K141Y-K256Q, K043T,K043T-K141Y-E156Q, N062L-Q217E, N062L-Q217L, N062L-S063G-Q217E,N062L-S063L, N062L-S063N-Q217L, N062S-Q217L, N062S-S063G, N062S-S063L,N062S-S063N-Q217L, N062S-S063R-Q217E, Q217L, S063G-Q217E, S063N-Q217E,S063R, S063R-Q217E, S063R-Q217L,), D099N-K141Y-K213Q, D099N-K141Y-K256Q,K043T, K043T-K141Y-E156Q, N062L-Q217E, N062L-Q217L, N062L-S063G-Q217E,N062L-S063L, N062S-Q217L, N062S-S063G, N062S-S063L, N062S-S063N-Q217L,Q217L, S063G-Q217E, S063N-Q217E, S063R, S063R-Q217E, and S063R-Q217L,wherein amino acid positions of the variant are numbered bycorrespondence with the sequence of SEQ ID NO:2. Such variants haveproteolytic activity. The invention includes a protease variant havingproteolytic activity and/or a PI value equal to or greater than 0.5 andequal to or less than 1.0 relative to BPN′-v36 in this assay, thevariant comprising an amino acid sequence having at least 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity toSEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acidsubstitutions selected from said group above, wherein amino acidpositions of the variant are numbered by correspondence with amino acidpositions of the SEQ ID NO:2 sequence. Also included are compositions,including, but not limited to, e.g., cleaning compositions, comprisingat least one such variant and methods for cleaning utilizing at leastone such protease variant as described in greater detail elsewhereherein.

The following BPN′-v36 variants were determined to have a PI value equalto or greater than 0.5 and less than 0.9 relative to BPN′-v36 in the BMImicroswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ IDNO:6) comprising at least one set of amino acid substitutions selectedfrom the group consisting of D099N, K141Y-E156Q, N062L-S063L-Q217L,N062L-S063N, N062L-S063N-Q217E, N062L-S063R, N062L-S063R-Q217L,N062S-Q217E, N062S-S063G-Q217E, N062S-S063G-Q217R, N062S-S063N-Q217R,S063G-S125A, D060G-Q217L,

D120N-K141Y-K213Q, K043T-D099N-D120N-K141Y, K043T-D099N-K141Y-K256Q,K043T-K237A, N062L-S063G-Q217R, N062L-S063G-S125A, N062L-S063L-Q217E,N062L-S063N-S125A-Q217L, N062S-Q217R, N062S-S063L-Q217E,N062S-S063R-Q217L, S063G-M222S, S063G-Q217R, D120N-E156Q-K256Q,K141Y-D197N, N062L-Q217R, N062L-S063G-Q217L-M222S, N062L-S063L-Q217R,N062L-S063N-Q217R, N062S-Q217G, N062S-S063G-Q217G,N062S-S063G-Q217L-M222L, N062S-S063G-S125A-Q217L, N062S-S063N-Q217E,Q217G, S033G-N062S-S063G, S063G-Q217G, S063G-Q217L-M222L,S063G-S125A-Q217R, S063L-Q217R, S063N-M222S, S063N-Q217R,S063N-S125A-Q217L, S063R-Q217R, S063R-S125A-Q217L, D099N-E156Q-K256Q,E156Q, K012T-D099N-K213Q, K012T-K256Q, K043T-D099N-K141Y-K213Q,K043T-E156Q, K141Y-K213Q, N062L-Q217G, N062L-Q217L-M222L,N062L-Q217L-M222S, N062L-S063G-M222S, N062L-S063G-Q217L-M222L,N062L-S063G-Q217R-M222S, N062L-S063N-Q217L-M222S, N062L-S063N-S125A,N062L-S063R-S125A, N062L-S125A, N062S-S063G-M222S,N062S-S063G-Q217G-M222S, N062S-S063G-S125A, N062S-S063N-Q217L-M222L,N062S-S063N-S125A-Q217L, N062S-S063R-Q217G, N062S-S063R-Q217L-M222S,Q217G-M222S, Q217L-M222S, Q217R, S033G-S063G-Q217R, S063G-Q217E-M222S,S063G-S125A-Q217G, S063L-Q217E, S063N-Q217G, S063N-Q217G-M222S,S063N-Q217L-M222S, S063R-Q217L-M222S, and S063R-S125A, wherein aminoacid positions of the variant are numbered by correspondence with thesequence of SEQ ID NO:2. Such variants have proteolytic activity. Theinvention includes a protease variant having proteolytic activity and/ora PI value equal to or greater than 0.5 and less than 0.9 relative toBPN′-v36 in this assay, the variant comprising an amino acid sequencehaving at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprisingat least one set of acid substitutions selected from said group above,wherein amino acid positions of the variant are numbered bycorrespondence with amino acid positions of the SEQ ID NO:2 sequence.Also included are compositions, including, but not limited to, e.g.,cleaning compositions, comprising at least one such variant and methodsfor cleaning utilizing at least one such protease variant as describedin greater detail elsewhere herein.

Part II

Methods for Making GG36 Cold Water Protease Variants

A variety of methods are known in the art that are suitable forgenerating modified polynucleotides of the invention that encode GG36cold water protease variants, including, but not limited to, forexample, site-saturation mutagenesis, scanning mutagenesis, insertionalmutagenesis, deletion mutagenesis, random mutagenesis, site-directedmutagenesis, and directed-evolution, as well as various otherrecombinatorial approaches. Non-limiting exemplary methods of making theSeries I GG36 cold water protease variants are provided in the sectionabove entitled “Vectors, Cells, and Methods for Making Protease VariantPolypeptides of the Invention.”

Example 19

For testing of enzyme activity in heat-inactivated detergents, workingsolutions of detergents are made from the heat inactivated stocks.Appropriate amounts of water hardness (e.g., 6 gpg or 12 gpg) and bufferare added to the detergent solutions to match the desired conditions.The solutions are mixed by vortexing or inverting the bottles. Thefollowing Table provides information regarding some of thecommercially-available detergents and test conditions used herein. Insome experiments, additional and/or other commercially availabledetergents find use in the following Examples.

TABLE 19-1 Laundry and Dish Washing Conditions T Region Form DoseDetergent* Buffer Gpg pH (° C.) Laundry (Heavy Duty Liquid and Granular)NA HDL 0.78 g/l   P&G TIDE ® 2X 5 mM HEPES 6 8.0 20 WE HDL 5.0 g/LHenkel 5 mM HEPES 12 8.2 40 PERSIL ™ WE HDG 8.0 g/L P&G ARIEL ® 2 mMNa₂CO₃ 12 10.5 40 JPN HDG 0.7 g/L P&G TIDE ® 2 mM Na₂CO₃ 6 10.0 20 NAHDG 1.0 g/L P&G TIDE ® 2 mM Na₂CO₃ 6 10.0 20 Automatic Dish Washing WEADW 3.0 g/L RB 2 mM Na₂CO₃ 21 10.0 40 CALGONIT ™ NA ADW 3.0 g/L P&G 2 mMNa₂CO₃ 9 10.0 40 CASCADE ®In some additional Examples, the following solutions find use:

TABLE 19-2 Working Detergent Solutions Temp Detergent Detergent (C.) g/LpH Buffer Gpg TIDE ® 2X Cold 16 0.98 8 5 mM HEPES 6 TIDE ® 2X Cold 320.98 8 5 mM HEPES 6 TIDE ® 2X Cold 16 0.98 7 5 mM MOPS 6Table 19-3 provides granular laundry detergent compositions produced inaccordance with the invention suitable for laundering fabrics.

TABLE 19-3 Granular Laundry Detergent Compositions and Their ComponentsDetergent Compositions Component 1 2 3 4 5 6 Linear 15 12 20 10 12 13alkylbenzenesulfonate with aliphatic carbon chain length C₁₁-C₁₂ Othersurfactants 1.6 1.2 1.9 3.2 0.5 1.2 Phosphate builder(s) 2 3 4 Zeolite 11 4 1 Silicate 4 5 2 3 3 5 Sodium Carbonate 2 5 5 4 0 3 Polyacrylate (MW4500) 1 0.6 1 1 1.5 1 Carboxymethyl cellulose 1 — 0.3 — 1.1 — (FinnfixBDA ex CPKelco) Celluclean ® (15.6 mg/g) 0.23 0.17 0.5 0.2 0.2 0.6 ColdWater Protease 0.23 0.17 0.05 0.2 0.03 0.1 variant* Stainzyme Plus ®0.23 0.17 0.5 0.2 0.2 0.6 (14 mg/g) Mannaway 4.0T (4 mg/g) 0.1 0.1 0.1Lipex 100T (18.6 mg/g) 0.2 0.1 0.3 Fluorescent Brightener(s) 0.16 0.060.16 0.18 0.16 0.16 Diethylenetriamine 0.6 0.6 0.25 0.6 0.6 pentaaceticacid or Ethylene diamine tetraacetic acid MgSO₄ 1 1 1 0.5 1 1 Bleach(es)and Bleach 6.88 6.12 2.09 1.17 4.66 activator(s) Ethoxylated thiophene0.002 0.001 0.003 0.003 — — Hueing Dye⁵ Direct Violet 9 ex Ciba 0.00060.0004 0.0006 Specialty Chemicals Sulfate/Citric Acid/ Balance to 100%Sodium Bicarbonate/ Moisture/perfumeIn Table 19-3, all enzyme levels expressed as % enzyme raw material,except for cold water protease variant (of this invention) which isexpressed as % of active protein added to the product. Table 19-4provides granular laundry detergent compositions suitable fortop-loading automatic washing machines (detergent compositions 7-9) andfront loading washing machines (detergent compositions 10-11). The GG36protease variant tested and/or BPN′ variant and/or cold water proteasevariant of the present invention is added separately to theseformulations.

TABLE 19-4 Granular Laundry Detergent Compositions and Their ComponentsComponent Detergent Composition Surfactants 7 8 9 10 11 C₁₆₋₁₇ Branchedalkyl sulfate 3.55 15.8 C₁₂₋₁₄ alkyl sulphate 1.5 Sodium linearalkylbenzenesulfonate 9.6 10.6 7.5 9 with aliphatic chain length C₁₁-C₁₂Sodium C_(14/15) alcohol ethoxy-3- 1.15 2.88 sulfate Sodium C_(14/15)alkyl sulphate 2.37 C_(14/15) alcohol ethoxylate with average 7 1.17 1moles of ethoxylation mono-C₈₋₁₀ alkyl mono-hydroxyethyl di- 0.45 methylquaternary ammonium chloride Di methyl hydroxyl ethyl lauryl 0.18ammonium chloride Zeolite A 13.9 4.7 0.01 2.9 1.8 Sodium Silicate1.6.ratio 4 0.2 4 4 Sodium Silicate 2.35.ratio 8 Citric Acid 2.5 1.4Sodium tripolyphosphate 5 Sodium Carbonate 24.1 30 16.9 24.4 21Nonanoyloxybenzenesuplhonate 5.78 2.81 0.96 Oxaziridinium-based bleachbooster 0.03 0.017 Tetrasodium S,S,- 0.2 ethylenediaminedisuccinateDiethylenetriamine penta (methylene 0.61 0.33 phosphonic acid),heptasodium salt Hydroxyethane dimethylene phosphonic 0.29 0.45 acidEthylene diamine tetraacetate 0.27 MgSO4 0.47 0.5994 0.782 SodiumPercarbonate 7 4.4 15.9 19.1 Tetra Acetyl Ethylene Diamine 3.3 4.6Sodium Perborate Monohydrate 1.2 Carboxymethyl cellulose 0.1 0.17 1.690.23 (e.g., Finnfix BDA ex CPKelco) Sodium Acrylic acid/maleic acid co-0.0236 3.8 2 2.5 polymer (70/30) Sodium polyacrylate (Sokalan PA30 CL) 40.84 Terephthalate polymer 0.23 Polyethylene glycol/vinyl acetate 0.890.89 0.91 random graft co polymer Photobleach- zinc phthalocyanine 0.0050.001 0.002 tetrasulfonate C.I. Fluorescent Brightener 260 0.11 0.150.04 0.23 0.15 C.I. Fluorescent Brightener 351 0.1 (Tinopal ® CBS) Sudssuppressor granule 0.25 0.07 0.04 Hydrophobically modified carboxy 0.0190.028 methyl cellulose (Finnifix ® SH-1) Bentonite 8.35 Miscellaneous(Dyes, perfumes, process Balance Balance Balance Balance Balance aids,moisture and sodium sulphate)

In Table 19-4, surfactant ingredients can be obtained from any suitablesupplier, including but not limited to BASF (e.g., LUTENSOL®), ShellChemicals, Stepan, Huntsman, and Clariant (e.g., PRAEPAGEN®). Zeolitecan be obtained from sources such as Industrial Zeolite. Citric acid andsodium citrate can be obtained from sources such as Jungbunzlauer.Sodium percarbonate, sodium carbonate, sodium bicarbonate and sodiumsesquicarbonate can be obtained from sources such as Solvay.Acrylate/maleate copolymers can be obtained from sources such as BASF.Carboxymethylcellulose and hydrophobically modified carboxymethylcellulose can be obtained from sources such as CPKelco. C.I. FluorescentBrightener 260 can be obtained from 3V Sigma (e.g., OPTIBLANC®,OPTIBLANC® 2M/G, OPTIBLANC® 2MG/LT Extra, or OPTIBLANC® Ecobright.Tetrasodium S,S-ethylenediamine disuccinate can be obtained from sourcessuch as Innospec. Terephthalate co-polymer can be obtained from Clariant(e.g., REPELOTEX SF 2). In addition, 1-Hydroxyethane -1,1-diphosphonicacid can be obtained from Thermphos. Oxaziridinium-based bleach boosterhas the following structure, where R1=2-butyloctyl, and was producedaccording to US 2006/0089284A1.

The enzymes NATALASE®, TERMAMYL®, STAINZYME PLUS®, CELLUCLEAN® andMANNAWAY® can be obtained from Novozymes. Zinc phthalocyaninetetrasulfonate can be obtained from Ciba Specialty Chemicals (e.g.,TINOLUX® BMC). Suds suppressor granule can be obtained from Dow Corning.In these detergent compositions, random graft copolymer is a polyvinylacetate grafted polyethylene oxide copolymer having a polyethylene oxidebackbone and multiple polyvinyl acetate side chains. The molecularweight of the polyethylene oxide backbone is about 6000 and the weightratio of the polyethylene oxide to polyvinyl acetate is about 40 to 60and no more than 1 grafting point per 50 ethylene oxide units.

Assays and Test Methods

This Example describes the various assays used and provides furtherdetails of Test Methods 4-6.

TCA Assay for Protein Content Determination in 96-well Microtiter Plates

For GG36 and GG36 variants, this assay was started using filtered B.subtilis culture supernatants from microtiter plates grown 2-3 days at37° C. with shaking at 250 rpm and humidified aeration. A fresh 96-wellflat bottom microtiter plate (MTP; Costar 9017 medium binding clearpolystyrene plate) was used for the assay. First, 100 μL/well of 0.25 NHCl was placed in each well. Then, 20-25 μL of filtered culturesupernatant were added and the solution was mixed on a table top mixer(e.g., Lab line Instruments, Titer plate shaker, model 4825) for 5-10seconds. The light scattering/absorbance at 405 nm was then determined,in order to provide the “blank” reading. For the “test” reading, 100μL/well of 30% (w/v) trichloroacetic acid (TCA) was added to each wellcontaining the mixture of HCl and culture supernatant, and the plate wasincubated for 10 minutes at room temperature. After briefly mixing thesolution on a table top mixer for no more than 2-3 sec, the lightscattering/absorbance at 405 nm was determined. The turbidity/lightscatter increase in the samples correlates to the total amount ofprecipitable protein in the culture supernatant. The calculations wereperformed by subtracting the “blank” reading (obtained after addition ofHCl only, no TCA) from the “test” reading (obtained after addition ofTCA, as described above) to provide a relative measure of the proteincontent in the samples. If desired, a standard curve can be created bycalibrating the TCA readings with AAPF assays of clones with knownconversion factors. However, the TCA results are linear with respect toprotein concentration from 50 to 500 parts per million (ppm) of protein(where 1 ppm corresponds to 1 mg/L) and can thus be plotted directlyagainst enzyme performance for the purpose of choosing variants withdesired performance.

AAPF Protease Assay in 96-well Microtiter Plates

In order to determine the protease activity of the serine proteasevariants, the hydrolysis ofN-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-p-nitroanilide(suc-AAPF-pNA) was measured. The reagent solutions used were: 100 mMTris/HCl, pH 8.6, containing 0.005% TWEEN®-80 (Tris dilution buffer);100 mM Tris buffer, pH 8.6, containing 1 mM CaCl₂ and 0.005% TWEEN®-80(Tris/Ca buffer); and 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stocksolution) (Sigma: S-7388). To prepare a suc-AAPF-pNA working solution, 1ml suc-AAPF-pNA stock solution was added to 100 ml Tris/Ca buffer andmixed well for at least 10 seconds. The assay was performed by adding 10μl of diluted protease solution to each well of a 96-well MTP,immediately followed by the addition of 190 μl of 1 mg/ml suc-AAPF-pNAworking solution. The solutions were mixed for 5 sec, and the absorbancechange in kinetic mode (25 readings in 5 minutes) was read at 405 nm inan MTP reader, at 25° C. The protease activity was expressed as AU(activity=ΔOD·min⁻¹ ml⁻¹).

Eglin C Inhibition Assay

As described herein, serine protease concentration and specific activitywas determined by titration with an inhibitor called eglin c. Eglin cfrom the leech Hirudo medicinalis is a tight-binding protein inhibitorof subtilisins and ASP protease (Heinz et al., Biochemistry, 31: 8755-66[1992]), and can therefore be used to measure protease enzymeconcentration, which in turn permits specific activity to be calculated.The gene for eglin c was synthesized and expressed in E. coli bystandard methods. Its properties and inhibitory potency were the same aseglin c purchased from Sigma.

(i) Concentration Determination of an Eglin C Stock Solution

A sample of Bacillus lentus subtilisin of known specific activity wasdiluted in 100 mM Tris buffer, pH 8.6, containing 1 mM CaCl₂ and 0.005%TWEEN®-80 (Tris/Ca buffer), to a concentration appropriate for AAPFprotease assay described above. Several dilutions of the eglin c stocksolution were also made in the Tris/Ca buffer. An aliquot of eachdiluted eglin c solution was mixed with an equal volume of the dilutedBacillus lentus subtilisin solution. An aliquot of the Tris/Ca bufferonly, without eglin c, was also mixed with an equal volume of thediluted Bacillus lentus subtilisin solution, in order to measureuninhibited subtilisin activity in the absence of eglin c. The mixedsolutions were incubated at room temperature for 15-30 minutes and theprotease activity of each sample was then measured by AAPF assaydescribed above. Using the known specific activity of Bacillus lentussubtilisin, the concentration of active protease in each sample wasdetermined. The concentration of eglin c in each sample was thencalculated based on the decrease of the observed protease activity ascompared to the uninhibited subtilisin sample that was mixed withTris/Ca buffer only (without eglin c). Thus, using the known dilutionsand volumes of the eglin c solutions, the concentration of eglin c inthe stock solution was determined.

(ii) Concentration and Specific Activity Determination of SubtilisinVariants

Samples of subtilisin variants were diluted in 100 mM Tris buffer, pH8.6, containing 1 mM CaCl₂ and 0.005% TWEEN®-80 (Tris/Ca buffer).Several dilutions of the eglin c stock solution of known concentrationwere also made in the Tris/Ca buffer. An aliquot of each diluted eglin csolution was mixed with an equal volume of a subtilisin variantsolution. The mixed solutions were incubated at room temperature for15-30 minutes and the protease activity of each sample was then measuredby AAPF assay. Using the observed decrease of the protease activity uponaddition of each eglin c sample and the known concentration of the eglinc, the concentration of the eglin c necessary for the completeinhibition of each subtilisin enzyme variant was calculated. Thisconcentration is equivalent to the enzyme concentration in the sample.An aliquot of the Tris/Ca buffer only, without eglin c, was also mixedwith each subtilisin variant sample and the protease activity in theabsence of eglin c was measured by AAPF assay. The specific activity ofthe subtilisin variants was then calculated using the enzymeconcentrations as determined above.

BMI Microswatch Assay of Test Methods 4-6

Blood milk and ink (BMI) stained microswatches (EMPA116) of 5 5millimeter circular diameter were obtained from CFT. In one method, theEMPA116 BMI fabric is pre-rinsed in water prior to cutting them into a96 well microtiter plate (Corning 3641), one microswatch per well. Inthe second method the EMPA 116 cloth is cut directly into a 96 wellmicrotiter plate (Corning 3641) where the swatches are then rinsed withtwo water washes. The rinses are carried out by adding 200 μl of Milli Qwater to each well/swatch and mixing them on a table top mixer (Lab lineinstruments, Titer plate shaker, model 4825) for 15 minutes at a settingof 7. The wash liquor is removed and 200 μl of water is added again tothe swatch for another 15 minute rinse. The wash water is removed andthe swatches are then air dried in the microtiter plate.

Detergent compositions 7-11 (Table 19-4) were diluted in Milli-Q(deionized) water to final working concentrations described in Table19-1. These detergents were buffered with 2 mM sodium carbonate, pH10.3. Additionally, a water hardness composition (3:1Ca:Mg.—CaCl₂:MgCl₂.6H₂O) was added to each detergent solution to thefinal concentration described in Table 19-5. The detergent solutionswere mixed at room temperature for 0.5 to 2 hours, centrifuged in 50 mLpolypropylene conical tubes at 3000×g for 5-10 minutes and were kept atroom temperature for the 32° C. assays or pre-equilibrated in anice-water bath for the 16° C. assays. Then, 190 μl of the desireddetergent solution was added to each well of the MTP containing BMImicroswatches. To this mixture, 5-15 μl of the diluted enzyme masterdilution solution were added, making the approximate concentration ofenzyme in the reaction 0.25-2 μg/ml. The enzyme master dilution solutionwas prepared from the filtered culture supernatants (see TCA assaydescribed above) at ˜2.5-20 μg/mL. The MTP was sealed with tape andplaced in the iEMS incubator/shaker (Thermo/Labsystems) pre-set at 16°C. in a refrigerator for 30 minutes or at 32° C. on the benchtop for 30minutes, with agitation at 1400 rpm. Following incubation under theappropriate conditions, 120-125 μl of the solution from each well wastransferred into a fresh MTP (Corning 9017). The new MTP containing 125μl of solution/well was read at 600 nm (with 5 sec mixing mode in theplate reader) using the MTP SpectraMax reader. Blank controls containinga microswatch and detergent without any enzyme were also included. Theabsorbance value obtained was corrected for the blank value (substratewithout enzyme), providing a measure of hydrolytic activity. For eachsample (variant) the performance index was calculated. The performanceindex compares the performance of the variant (measured value) and thestandard enzyme (theoretical value) at the same protein concentration.In addition, the theoretical values can be calculated, using theparameters of a performance dose response curve of the standardprotease. A performance index (PI) that is greater than 1 (PI>1)identifies a better variant as compared to the standard (e.g.,wild-type), while a PI of 1 (PI=1) identifies a variant that performsthe same as the standard, and a PI that is less than 1 (PI<1) identifiesa variant that performs worse than the standard. Thus, the PI identifieswinners, as well as variants that are less desirable for use undercertain circumstances.

TABLE 19-5 Final Detergent, Water Hardness, and Buffer ConcentrationsUsed for BMI Microswatch Assays Final Sodium Final Detergent Final WaterCarbonate Buffer Detergent Concentration Hardness* ConcentrationComposition (g/L) (gpg) (mM) 7 0.808 6 2 8 1 3 2 9 2.3 12 2 10 5.9 12 211 8.3 12 2LAS/EDTA Stability Assay

The stability of protease variants in the presence of a representativeanionic surfactant (LAS=linear alkylbene sulfonate, sodiumdodecylbenzenesulfonate-DOBS) and di-sodium EDTA is measured afterincubation under defined conditions and the residual activity isdetermined using the AAPF assay described above. The reagents used weredodecyllbenzene sulfonate, sodium salt (DOBS; Sigma No. D-2525),TWEEN®-80 (Sigma No. P-8074), di-sodium EDTA (Siegfried Handel No.164599-02), HEPES (Sigma No. H-7523), unstressed buffer: 50 mM HEPES(11.9 g/l)+0.005% TWEEN®-80, pH 8.0, Stress buffer: 50 mM HEPES (11.9g/l), 0.1% (w/v) DOBS (1 g/l), 10 mM EDTA (3.36 g/l), pH 8.0, referenceprotease and protease variant culture supernatants, containing 200-400μg/ml protein. The equipment used is V- or U-bottom MTP as dilutionplates (Greiner 651101 and 650161 respectively), F-bottom MTP (Corning9017) for unstress and LAS/EDTA buffer as well as for suc-AAPF-pNAplates, Biomek FX (Beckman Coulter), Spectramax Plus 384 MTP Reader(Molecular Devices), and iEMS Incubator/Shaker (Thermo/Labsystems).

The iEMS incubator/shaker (Thermo/Labsystems) is set at 29° C. Culturesupernatants were diluted into plates containing unstress buffer to aconcentration of ˜25 ppm (master dilution plate). For the assay, 20 μlof sample from the master dilution plate is added to plates containing180 μl unstress buffer to give a final incubation concentration of 2.5ppm. The contents were mixed and kept at room temperature and the AAPFassay is performed on this plate. In addition, 20 μl of sample from themaster dilution plate is also added to plates containing 180 μl stressbuffer (50 mM HEPES (11.9 g/l), 0.1% (w/v) DOBS (1 g/l), 10 mM EDTA(3.36 g/l), pH 8.0). The solutions were mixed and immediately placed in29° C. iEMS shaker for 30 min at 400 rpm. Following 30 minutes ofincubation, the AAPF assay is performed on the stress plate. Thestability of the samples is determined by calculating the ratio of theresidual and initial AAPF activity as follows: Residual Activity(%)=[mOD·min−1 stressed]*100/ [mOD·min−1 unstressed].

The final detergent, water hardness and buffer concentrations aredetermined based on the assay system to be used (e.g., North American,Japanese, Western European, or Central European conditions). In someaspects, the stain removal performance of the protease variants isdetermined in commercially available detergents. Heat inactivation ofcommercial detergent formulas serves to destroy the enzymatic activityof any protein components while retaining the properties ofnon-enzymatic components. Thus, this method is suitable for preparingcommercially purchased detergents for use in testing the enzyme variantsof the present invention.

Baked Egg Microtiter Assay

For this assay, 96-well baked egg yolk substrate plates are preparedfrom chicken egg yolks. Chicken egg yolks are separated from the whites,released from the membrane sac, and diluted 20% (vol/weight) withMilli-Q water. The diluted yolk is stirred for 15 min at roomtemperature using a magnetic stirrer. Five μL are carefully pipettedinto the center of each well of a 96-well V-bottom plate (Costar #3894)using an 8-channel pipette. The plates are baked at 90° C. for 1 hourand cooled at room temperature. The baked egg yolk substrate plates arestored at room temperature and used within one week of preparation.Automatic dish detergents are prepared as described herein andpre-heated to 50° C. A 190 μL aliquot of detergent is added to each wellof the 96-well plate using an 8-channel pipette. Ten μL of dilutedenzyme is added to each well using a 96-channel pipetting device. Theplate is carefully sealed with an adhesive foil sealer and incubated at50° C. with shaking for 30 min. 120 μL of the reaction mixture istransferred to a new 96-well flat-bottom plate, and the absorbance/lightscattering is determined at 405 nm. The absorbance/light scattering at405 nm is proportional to egg yolk removal.

Performance Index

The performance index compares the performance of the variant (measuredvalue) and the standard enzyme (theoretical value) at the same proteinconcentration. In addition, the theoretical values can be calculated,using the parameters of a performance dose response curve of thestandard protease. Various terms set forth below are used to describethe variant: non-deleterious variants have a PI>0.05; deleteriousvariants have a PI=0.05; combinable variants are those for which thevariant has performance index values greater than or equal to 0.2 for atleast one property, and >0.05 for all properties. Combinable variantsare those that can be combined to deliver proteins with appropriateperformance indices for one or more desired properties. These data finduse in engineering any subtilisin/subtilase. Even if the subtilase to beengineered has an amino acid different from that of subtilisin GG36 atparticular positions, these data find use in finding substitutions thatwill alter the desired properties by identifying the best choices forsubstitutions, including substitutions to the GG36 wild type amino acid.

Example 20 Generation of GG36 Single Mutants Using Site EvaluationLibraries (SELs)

The construction of GG36 SELs described in this example was performed byGENEART using their proprietary methods and technology platform for geneoptimization, gene synthesis, library generation and analysis (WO2004/059556A3, European Patent Nos. 0 200 362 and 0 201 184; and U.S.Pat. Nos. 4,683,195, 4,683,202 and 6,472,184). The GG36 SELs wereproduced at positions pre-selected by the inventors using the pHPLT-GG36B. subtilis expression plasmid (see FIG. 6). This B. subtilis expressionplasmid contains the GG36 expression cassette shown below, the B.licheniformis LAT promoter (Plat), and additional elements from pUB110(McKenzie et al., Plasmid, 15:93-103, 1986) including a replicase gene(reppUB), a neomycin/kanamycin resistance gene (neo) and a bleomycinresistance marker (bleo) (FIG. 4 in U.S. Pat. No. 6,566,112). ThepHPLT-GG36 plasmid map is provided at FIG. 6. The GG36 expressioncassette sequence is provided below.

The DNA sequence of GG36 (the signal sequence is shown in lower caseletters, propeptide in lower case, underlined text, and GG36 maturesequence in uppercase letters) is provided below:

(SEQ ID NO: 756) GtgagaagcaaaaaattgtggatcgtcgcgtcgaccgcactactcatttctgttgctttcagttcatcgatcgcatcggctgctgaagaagcaaaagaaaaatatttaattggctttaatgagcaggaagctgtcagtgagtttgtagaacaagtagaggcaaatgacgaggtcgccattctctctgaggaagaggaagtcgaaattgaattgcttcatgaatttgaaacgattcctgttttatccgttgagttaagcccagaagatgtggacgcgcttgagctcgatccagcgatttcttatattgaagaggatgcagaagtaacgacaatgGCGCAATCAGTGCCATGGGGAATTAGCCGTGTGCAAGCCCCAGCTGCCCATAACCGTGGATTGACAGGTTCTGGTGTAAAAGTTGCTGTCCTCGATACAGGTATTTCCACTCATCCAGACTTAAATATTCGTGGTGGCGCTAGCTTTGTACCAGGGGAACCATCCACTCAAGATGGGAATGGGCATGGCACGCATGTGGCCGGGACGATTGCTGCTTTAAACAATTCGATTGGCGTTCTTGGCGTAGCGCCGAGCGCGGAACTATACGCTGTTAAAGTATTAGGGGCGAGCGGTTCAGGTTCGGTCAGCTCGATTGCCCAAGGATTGGAATGGGCAGGGAACAATGGCATGCACGTTGCTAATTTGAGTTTAGGAAGCCCTTCGCCAAGTGCCACACTTGAGCAAGCTGTTAATAGCGCGACTTCTAGAGGCGTTCTTGTTGTAGCGGCATCTGGAAATTCAGGTGCAGGCTCAATCAGCTATCCGGCCCGTTATGCGAACGCAATGGCAGTCGGAGCTACTGACCAAAACAACAACCGCGCCAGCTTTTCACAGTATGGCGCAGGGCTTGACATTGTCGCACCAGGTGTAAACGTGCAGAGCACATACCCAGGTTCAACGTATGCCAGCTTAAACGGTACATCGATGGCTACTCCTCATGTTGCAGGTGCAGCAGCCCTTGTTAAACAAAAGAACCCATCTTGGTCCAATGTACAAATCCGCAATCATCTAAAGAATACGGCAACGAGCTTAGGAAGCACGAACTTGTATGGAAGCGGACTTGTCAATGCAGAAGCTGCAACTCGTTAA

The protein sequence of GG36 (the signal sequence is shown in lower caseletters, propeptide in lower case, underlined text, and GG36 matureprotease sequence in uppercase letters) is provided below:

(SEQ ID NO: 757) vrskklwivastallisvafsssiasaaeeakekyligfneqeavsefveqveandevailseeeeveiellhefetipvlsvelspedvdaleldpaisyieedaevttmAQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR.

The method of mutagenesis was based on the codon-specific mutationapproach in which all possible amino acid substitutions aresimultaneously created at a specific codon of interest using forward andreverse mutagenesis primers that contain a degenerate codon, NNS ((A,C,Tor G), (A,C,T or G), (C or G)) at the site of interest. To constructeach of the GG36 SELs, three PCR reactions were performed: twomutagenesis reactions (primary PCR1 and PCR2) to introduce the mutatedcodon of interest in the mature GG36 DNA sequence using the NNS forwardand reverse mutagenesis primers (25-45 nucleotides long), and a thirdreaction to fuse the two mutagenesis PCR products together to constructthe pHPLT-GG36 expression vector having the desired mutated codons inthe mature GG36 sequence.

The primer sequences used in this Example are provided below:

TABLE 20-1 Primers Sequence Primer Name CGCGCTTGAGCTCGATCCAGCGATTTCSacI-Fw (SEQ ID NO: 758) GTCTCCAAGCTTTAACGAGTTGCAG HindIII-Rv(SEQ ID NO: 759) GCAATTCAGATCTTCCTTCAGGTTATGACC pHPLT-BglII-Fw(SEQ ID NO: 760) GCATCGAAGATCTGATTGCTTAACTGCTTC pHPLT-BglII-Rv(SEQ ID NO: 761)

The Phusion High-Fidelity DNA Polymerase (Finnzymes catalog no. F-530L)was used for all PCRs, and the reactions were executed according tomanufacturer's protocols that were supplied with the polymerase. Inparticular, for primary PCR 1, 1 μL (10 μM) of each of thepHPLT-BglII-Fw primer and a NNS reverse mutagenesis primer were used,and for primary PCR 2, 1 μL (10 μM) of the pHPLT-BglII-Rv primer and aNNS forward mutagenesis primer were used. Each reaction also included 1μL of the pHPLT-GG36 plasmid template DNA (0.1-1 ng/μL).An MJ ResearchPTC-200 Peltier thermal cycler was used for the PCRs. The reactionsyielded two fragments of approximately 2 to 3 kb having approximately 30nucleotide overlap surrounding the GG36 codon of interest. The fragmentsobtained were fused in a third PCR similar to the ones described aboveusing 1 μL of primary PCR 1 reaction mix, 1 μL of primary PCR 2 reactionmix and 1 μL (10 μM) of each of the forward and reverse SacI-Fw andHindIII-Rv primers. The amplified linear 859 bp fragment encoding theGG36 variant gene was purified (using QIAGEN® Qiaquick PCR purificationkit) and digested with the SacI and HindIIII restriction enzymes tocreate cohesive ends on both sides of the fusion fragment. About 50 ngof plasmid pHPLT-GG36 was also purified after digestion with SacI andHindIIII, resulting in a 3.9 kb vector backbone fragment. The digestedvector fragment was ligated with 50 ng of the digested 859 bp fragmentencoding the variant enzyme using the T4 DNA ligase (Invitrogen)following the manufacturer's protocol for cloning of cohesive ends.Subsequently, the ligation mixture was used to transform B. subtiliscells (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE::[xylR,pxylA-comK])as described (WO 2002/014490).

To express the variant proteins for further biochemical analyses, the B.subtilis strains carrying the GG36 variant plasmids were inoculated intomicrotiter plates containing 150 μl Luria broth medium supplemented with10 μg/ml neomycin. Plates were grown overnight at 37° C. with 300 rpmshaking and 80% humidity using Enzyscreen lids for microtiter plates(Enzyscreen). Ten microliters from the overnight culture plate were usedto inoculate a new microtiter plate containing 190 μl of MBD medium (aMOPS based defined medium) with 10 ug/ml neomycin. MBD medium wasprepared essentially as known in the art (see Neidhardt et al., J.Bacteriol. 119:736-747 [1974]), except that NH₄Cl, FeSO₄, and CaCl₂ wereomitted from the base medium, 3 mM K₂HPO₄ was used, and the base mediumwas supplemented with 60 mM urea, and 100 ml of a solution made of 210g/L glucose, and 350 g/L maltodextrin. The micronutrients were made upas a 100× stock solution containing in one liter, 400 mg FeSO₄.7H₂O, 100mg MnSO₄.H₂O, 100 mg ZnSO₄.7H₂O, 50 mg CuCl₂.2H₂O, 100 mg CoCl₂.6H₂O,100 mg NaMo₄.2H₂O, 100 mg Na₂B₄O₇.10H₂O, 10 ml of 1M CaCl₂, and 10 ml of0.5 M sodium citrate. The MBD medium containing microtiter plates weregrown for 68 hours at 37° C., 300 rpm, and 80% humidity using Enzyscreenlids (Enzyscreen) for determining protein expression. The next day,cultures were filtered through a micro-filter plate (0.22 μm; Millipore)and the resulting filtrate was used for biochemical analysis. The TCAand BMI microswatch assays for the detergent compositions 7-11 werecarried out as described in Example 19. Performance indices were alsocalculated as described under the BMI assay of Test Methods 4-6 asdescribed in detail in Example 19, and they are shown in Table 20-2relative to GG36. In the following Tables, the detergent compositions(“Det.”) correspond to those shown in Table 19-4, above. Also, asindicated, the amino acid position is listed according to BPN′numbering.

TABLE 20-2 Single Variants of GG36 with Performance Indices of at Least0.2 Relative to GG36 in Either TCA or BMI Microswatch Cleaning at 16° C.in Detergents 7-11. GG36 Amino Acid Position (BPN′ Numbering) WT ResidueMutant Residue 1 A R 2 Q A 2 Q R 2 Q S 2 Q M 2 Q W 3 S R 4 V R 4 V S 4 VC 8 I A 9 S W 9 S F 9 S A 10 R A 10 R M 10 R S 10 R H 12 Q F 12 Q R 14 PF 14 P K 14 P Q 15 A R 15 A F 16 A S 17 H R 17 H F 17 H M 18 N R 18 N K20 G R 20 G K 20 G F 22 T R 22 T Q 22 T L 22 T V 22 T W 22 T Y 22 T A 23G A 23 G S 23 G F 24 S R 24 S W 24 S H 24 S L 24 S Q 24 S F 25 G R 25 GF 25 G V 26 V F 27 K R 27 K L 27 K V 27 K F 28 V A 28 V E 28 V N 29 A T30 V E 31 L F 33 T S 33 T G 33 T D 34 G P 35 I M 36 S T 36 S F 36 S R 38T R 38 T F 38 T L 40 P H 40 P W 40 P R 40 P N 40 P T 40 P L 42 L I 43 NR 43 N A 43 N S 43 N W 43 N F 43 N I 43 N D 43 N M 45 R T 46 G R 48 A R50 F C 51 V W 51 V F 51 V H 52 P F 52 P N 52 P E 55 P Y 57 T R 59 Q A 59Q F 59 Q R 60 D P 60 D A 60 D Q 62 N Q 62 N E 63 G S 63 G A 63 G M 63 GV 63 G T 63 G H 63 G Q 63 G I 63 G D 63 G E 63 G P 64 H F 64 H T 68 V A68 V C 69 A N 69 A T 69 A W 69 A P 71 T G 72 I C 74 A C 75 L R 75 L A 75L E 75 L F 78 S R 78 S I 78 S N 79 I Q 79 I W 81 V R 82 L R 82 L T 82 LM 82 L F 82 L V 85 A M 86 P W 86 P I 86 P L 89 E P 89 E W 89 E T 89 E I89 E H 89 E V 89 E F 89 E L 89 E W 89 E G 91 Y F 91 Y N 92 A F 94 K N 99S F 99 S T 99 S M 99 S G 99 S P 100 G I 100 G S 100 G N 100 G Q 101 S N101 S G 101 S T 101 S A 101 S D 101 S F 101 S D 101 S E 101 S P 102 G A102 G N 102 G T 102 G E 102 G H 103 S N 103 S G 103 S D 104 V L 104 V I104 V E 104 V D 105 S T 105 S Q 105 S E 106 S V 106 S G 106 S T 106 S A106 S E 106 S D 106 S F 107 I F 107 I M 108 A I 108 A G 109 Q M 111 L V111 L I 112 E V 112 E L 112 E Q 114 A G 115 G R 115 G K 116 N L 116 N A116 N K 117 N F 118 G I 118 G R 119 M C 120 H A 120 H F 120 H R 121 V E121 V F 123 N G 123 N E 124 L S 128 S N 128 S M 128 S H 128 S Q 128 S I128 S F 128 S L 128 S D 129 P E 132 S A 132 S E 138 A G 144 S R 147 V L148 L I 158 A E 159 G C 159 G E 160 S D 166 S E 166 S D 167 Y W 175 M V177 V C 181 D A 182 Q R 183 N D 183 N R 183 N I 183 N F 183 N M 185 N I185 N E 185 N V 186 R H 186 R K 188 S R 188 S E 188 S D 192 Y W 192 Y H194 A V 194 A F 194 A E 197 D F 198 I L 198 I F 203 V E 203 V C 208 T S209 Y N 209 Y S 209 Y F 209 Y T 209 Y H 209 Y L 209 Y G 209 Y E 210 P V210 P R 210 P L 211 G R 211 G Q 212 S I 212 S F 212 S M 213 T A 214 Y F215 A F 215 A N 215 A H 215 A E 215 A D 216 S F 216 S A 217 L E 217 L N217 L D 218 N P 218 N E 218 N D 224 T A 224 T G 227 V I 230 A E 231 A I231 A C 233 L C 234 V F 235 K F 236 Q N 236 Q F 238 N R 238 N K 238 N L239 P R 239 P S 239 P R 239 P H 239 P N 239 P K 239 P T 239 P F 239 P G240 S R 241 W R 242 S R 242 S L 243 N R 243 N F 244 V R 246 I S 248 N I248 N V 248 N R 249 H R 249 H T 250 L I 251 K R 251 K S 252 N R 252 N F252 N H 252 N I 253 T R 253 T F 253 T I 254 A C 256 S N 258 G R 260 T V260 T I 262 L H 262 L D 263 Y F 265 S F 267 L N 267 L M 267 L V 269 N R269 N I 270 A C 271 E T 271 E V 271 E L 271 E H 271 E F 271 E P 271 E A271 E M 271 E I 272 A F 272 A R 273 A I 273 A F 274 T G

TABLE 20-3 GG36 Single Variants with Performance Indices of at Least 1.5Relative to GG36 BMI Microswatch Cleaning at 32° C. in Detergent 7. GG36Variant N62E A158E G159E

TABLE 20-4 GG36 Single Variants with Performance Indices of at least 1.2Relative to GG36 BMI Microswatch Cleaning at 32° C. in Detergent 10.GG36 Variant A1R S78R V244R N269R E271L

Example 21 Construction and Cleaning Performance of the NHJ1 and WCE1Sets of GG36 Variants

The NHJ1 and WCE1 set of GG36 variants described herein were constructedat DNA 2.0, Inc., using the pHPLT-GG36 B. subtilis expression plasmiddescribed above (FIG. 6). The variants were expressed in B. subtiliscells (genotype: ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo) as describedin Example 20, and were further characterized using the TCA assay forprotein content determination, LAS/EDTA stability assay, and BMImicroswatch cleaning assay as described in Example 19. These results areshown in Tables 21-1 and 21-2. In the following Tables, the detergentcompositions (“Det.”) correspond to those shown in Table 19-4, above.Also, as indicated, the amino acid position is listed according to BPN′numbering.

TABLE 21-1 NHJ1 Variants with Performance Indices of at least 0.25Relative to GG36 in Any One of TCA, LAS/EDTA Stability, or BMIMicroswatch Cleaning at 16° C. in Detergents 7, 8 or 9. GG36 Variant(BPN′ numbering) N062E-A158E S101A-H249R A016S-S101A S103G-A158ES188D-H249R E089P-S101A S128N-A158E V104L-H249R N062E-S103G A016S-A158EY209E-H249R T022A-S103G V104L-A158E T022A-H249R A016S-S103G E089P-A158ES128N-H249R S101A-S103G L111V-A158E S103G-H249R E089P-S103G T022A-A158EE089P-H249R N062E-S128N S101A-A158E T022A-L111V A016S-S128N L148I-A158ES101A-L111V T022A-S128N P129E-A158E A016S-L111V S101A-S128N T022A-E089PV104L-L111V V104L-S128N A016S-E089P N062E-L111V E089P-S128N N062E-E089PS103G-L111V S103G-S128N N062E-E271F E089P-L111V L111V-S128N A158E-E271FA016S-L148I L111V-S188D R186H-E271F N062E-L148I N062E-S188D P129E-E271FT022A-L148I A016S-S188D L111V-E271F P129E-L148I L148I-S188D Y209E-E271FV104L-L148I T022A-S188D A016S-E271F S103G-L148I S128N-S188D S188D-E271FS128N-L148I S101A-S188D T022A-E271F S101A-L148I V104L-S188D G159E-E271FE089P-L148I E089P-S188D V104L-E271F L111V-L148I P129E-S188D S101A-E271FA016S-N062E G159E-S188D E089P-E271F T022A-N062E R186H-S188D S128N-E271FN062E-P129E S103G-S188D S103G-E271F T022A-P129E A158E-S188D L148I-E271FS128N-P129E A016S-T022A H249R-E271F A016S-P129E A016S-V104L N062E-G159ES101A-P129E T022A-V104L A016S-G159E V104L-P129E S101A-V104L S128N-G159EE089P-P129E N062E-V104L L148I-G159E S103G-P129E S103G-V104L L111V-G159EL111V-P129E E089P-V104L E089P-G159E N062E-R186H G159E-Y209E T022A-G159ES128N-R186H L111V-Y209E P129E-G159E S101A-R186H S101A-Y209E S103G-G159ET022A-R186H A016S-Y209E V104L-G159E A016S-R186H S128N-Y209E A158E-G159EA158E-R186H L148I-Y209E S101A-G159E E089P-R186H P129E-Y209E A158E-H249RP129E-R186H N062E-Y209E L111V-H249R G159E-R186H T022A-Y209E P129E-H249RS103G-R186H S103G-Y209E N062E-H249R V104L-R186H A158E-Y209E A016S-H249RL111V-R186H S188D-Y209E R186H-H249R L148I-R186H V104L-Y209E L148I-H249RN062E-S101A E089P-Y209E G159E-H249R T022A-S101A R186H-Y209E

TABLE 21-2 WCE1 Variants with Performance Indices of at least 0.2Relative to GG36 in Any One of TCA, LAS/EDTA Stability, or BMIMicroswatch Cleaning at 16° C. in Detergents 10 or 11. GG36 Variant(BPN′ Numbering) N018R-W241R G115R-S212F A001R-L082R G020R-W241RG020R-S212F S078R-L082R S024R-W241R S009A-S212F G020R-L082R S009A-W241RN043R-S212F T022R-L082R G020R-W241R S078R-S212F V004R-L082R V004R-W241RL082R-S212F N043R-L082R N043R-W241R S009A-S078R N043R-H249R S078R-W241RG020R-S078R G020R-H249R T022R-W241R S024R-S078R V004R-H249R G115R-W241RT022R-S078R N018R-H249R A001R-W241R N018R-S078R S009A-H249R S212F-W241RV004R-S078R S212F-H249R L082R-W241R A001R-S078R T022R-H249R N018R-V244RN043R-S078R S024R-H249R S024R-V244R T022R-S024R G115R-H249R S078R-V244RG020R-S024R A001R-H249R G020R-V244R N018R-S024R L082R-H249R S212F-V244RA001R-S024R S242R-H249R S009A-V244R V004R-S024R W241R-H249R L082R-V244RS009A-S024R V244R-H249R A001R-V244R V004R-S009A S078R-H249R N043R-V244RA001R-S009A N018R-G115R T022R-V244R S242R-N269R G020R-G115R V004R-V244RS024R-N269R T022R-G115R G115R-V244R G020R-N269R S078R-G115R W241R-V244RT022R-N269R S009A-G115R S242R-V244R H249R-N269R V004R-G115R A001R-V004RS212F-N269R A001R-G115R S009A-T022R N043R-N269R L082R-G115R N018R-T022RV244R-N269R N043R-G115R G020R-T022R A001R-N269R S024R-G115R V004R-T022RN018R-N269R S009A-G020R A001R-T022R S078R-N269R N018R-G020R S024R-S242RS009A-N269R V004R-G020R N018R-S242R G115R-N269R A001R-G020R V004R-S242RW241R-N269R S009A-E271L G020R-S242R V004R-N269R G020R-E271L S212F-S242RL082R-N269R S024R-E271L L082R-S242R N018R-N043R V244R-E271L S078R-S242RG020R-N043R W241R-E271L A001R-S242R V004R-N043R N043R-E271L S009A-S242RT022R-N043R T022R-E271L T022R-S242R S009A-N043R H249R-E271L G115R-S242RA001R-N043R S212F-E271L N043R-S242R S024R-N043R G115R-E271L W241R-S242RS009A-N018R S242R-E271L N018R-S212F V004R-N018R S078R-E271L T022R-S212FA001R-N018R V004R-E271L V004R-S212F S024R-L082R N269R-E271L S024R-S212FS009A-L082R A001R-E271L A001R-S212F N018R-L082R N018R-E271L L082R-E271L

Example 22 Construction and Cleaning Performance of NHJ4 Set of GG36Variants

The NHJ4 set of GG36 variants described in Table 22-1 below wereconstructed using the pHPLT-GG36 B. subtilis expression plasmid (FIG. 6)using PCR fusion or the QUIKCHANGE® Multi Site-directed mutagenesis kit(“QCMS kit”; Stratagene) as described below.

a) Construction of NHJ4 Variants by QUIKCHANGE® Multi Site-DirectedMutagenesis

Variants created using the QUIKCHANGE® Multi Site-Directed Mutagenesisare shown in Table 22-1. The parent plasmid pHPLT-GG36 (template DNA)was methylated using two micrograms of DNA and Dam methylase (NEB),according to the manufacturer's instructions. Site-directed mutants weremade by a QuikChange® Multi Site-Directed Mutagenesis Kit (“QCMS kit”;Stratagene) following the manufacturer's protocol. The followingmutations were introduced in the parent plasmid

S101A-S103G-V104L, G159E, T22A, Y209E, E271F, S101A, S103G, L111V,S128N, N62E, and S188D, For efficient transformation of B. subtilis, DNAfrom the QCMS reaction mixtures was amplified by rolling circleamplification (RCA) using the Illustra Templiphi kit (GE Healthcare) andthe reaction was performed according to the manufacturer's protocol. Onemicroliter of ten-fold diluted amplified DNA was used to transform 50 μLof competent B. subtilis cells (genotype: ΔaprE, ΔnprE,amyE::xylRPxylAcomK-phleo). The transformation mixture was shaken at 37°C. for 1 hour. Ten microliter aliquots of the transformation mixturewere plated on skim milk (1.6%) Luria agar plates supplemented with 10μg/ml of neomycin (Teknova). Subsequently, the colonies with halos wereinoculated in 120 μl of LB media containing 10 μg/mL neomycin forplasmid DNA extraction (QIAprep Spin Miniprep kit, Qiagen). Theextracted plasmids were sequenced to confirm the presence of the desiredmutations.

b) Construction of NHJ4 Variants by Extension PCR

Ten combinatorial mutants of GG36 were created by extension PCR. Thelist of mutations introduced in the GG36 gene contained in the pHPLTplasmid were T22A, N62E, S103G, S103G-L111V, S101G-S103A-V104I,S101A-S103G-V104L, S101A, S128N, G159D, G159E, Y209E, and L111V. Tocreate each mutant, PCR fragments containing the desired mutations wereamplified using mutagenic primers as well as forward and reverse primersto amplify the entire GG36 variant. Each PCR amplification reactioncontained 30 pmol of each mutagenic primer and 100 ng of the DNAtemplate, pHPLT-GG36 plasmid Amplifications were carried out using VentDNA polymerase (NEB). The PCR reaction (20 μL) was initially heated at95° C. for 2.5 min followed by 30 cycles of denaturation at 94° C. for15 sec., annealing at 55° C. for 15 sec. and extension at 72° C. for 1min. Following amplification, 2 to 4 PCR fragments for each variant weregel-purified, using a QIAGEN® gel-band purification kit and mixed (50 ngof each fragment). These mixtures served as DNA templates for theextension PCR to generate the full-length gene fragments. The PCRconditions were same as described above, except the extension phase,which was carried out at 72° C. for 2 min. The full-length DNA fragmentwas gel-purified using a QIAGEN® gel-band purification kit, digestedwith the BamHI and HindIII restriction enzymes and ligated with thepHPLT-GG36, which was digested with the same restriction enzymes. Onemicroliter of the ligation mixtures was amplified using rolling circleamplification by Illustra Templiphi kit according to the manufacturer'sinstructions (GE Healthcare) to generate multimeric DNA fortransformation into Bacillus subtilis. Products of the rolling circleamplification were diluted 100-times and used to transform B. subtiliscells (genotype: ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). An aliquot ofthe transformation mix was plated on LB plates containing 1.6% skim milkand 10 μg/mL neomycin and incubated overnight at 37° C. Subsequently,the colonies with halos were inoculated in 120 μl of Luria broth mediumcontaining 10 μg/mL neomycin for plasmid DNA extraction (QIAprep SpinMiniprep kit, Qiagen). The extracted plasmids were sequenced to confirmthe presence of the desired mutations. Variants created by the extensionPCR are shown in Table 22-1.

To express the NHJ4 set of variant proteins for further biochemicalanalyses, the B. subtilis strains carrying the variant plasmids wereinoculated into microtiter plates containing 150 μl Luria broth mediumsupplemented with 10 μg/ml neomycin. The cultures were grown up forprotein expression as described in Example 20, and they were filteredthrough a micro-filter plate (0.22 μm; Millipore) also as described inExample 20. The resulting filtrate was used for biochemical analysis.The eglin c inhibition assay for protein content determination and BMImicroswatch assays tested in various detergents were carried out asdescribed in Example 19. Performance indices are also calculated asdescribed under the BMI assay description of Test Methods 4-6 furtherdetailed in Example 19. Table 22-1 provides information regarding thesemultiple mutation variants and the results obtained for them. The PIvalues are relative to GG36. In the following Tables, the detergentcompositions (“Det.”) correspond to those shown in Table 19-4, above.Also, as indicated, the amino acid position is listed according to BPN′numbering.

TABLE 22-1 NHJ4 Multiple Mutation Variants with BMI Cleaning PerformanceIndices of at Least 0.2 Relative to GG36 in Detergents 7, 8 or 9 at 16°C. Variant Name Created by Mutations, (BPN′ Numbering) GG36 NHJ4-1Extension PCR S101G S103A V104I NHJ4-10 Extension PCR T22A S101A Y209ENHJ4-11 Extension PCR S103G L111V G159E NHJ4-12 Extension PCR T22A S103GG159E NHJ4-13 QCMS T22A L111V G159E NHJ4-14 QCMS T22A S128N E271F Y209ENHJ4-15 QCMS T22A S103G L111V NHJ4-16 QCMS N62E L111V S128N NHJ4-17 QCMST22A L111V S128N NHJ4-18 Extension PCR T22A N62E L111V NHJ4-19 QCMSS101A S103G V104L S188D NHJ4-2 Extension PCR S101G S103A V104I G159DNHJ4-20 Extension PCR S101A S103G V104L S128N NHJ4-24 QCMS T22A S101AG159E NHJ4-3 Extension PCR S101A S103G V104L NHJ4-4 Extension PCR S101AS103G V104L G159E NHJ4-5 Extension PCR T22A S101A S103G V104L NHJ4-6QCMS S101A S103G V104L Y209E NHJ4-7 QCMS T22A Y209E E271F NHJ4-8 QCMST22A S101A E271F NHJ4-9 QCMS S101A Y209E E271F

Example 23 Construction and Cleaning Performance of NHJ3 Set of GG36Variants

The NHJ3 set of variants described herein are based on a variant of GG36(referred to as GG36-9) containing the following mutations: S101G,S103A, V104I, G159D, A232V, Q236H, Q245R, N248D, and N252K (BPN′numbering). These variants were created using the QUIKCHANGE® LightningSite-Directed Mutagenesis Kit (QCLDS kit; Stratagene), with the pRA68plasmid (see FIG. 7) as the DNA template. Plasmid pRA68 was derived fromthe pBN3 vector (see Babe et al., Biotech. Appl. Biochem. 27:117-124[1998]).

The DNA sequence of GG36-9 variant (the signal sequence is shown inlower case letters, propeptide in lower case, underlined text, andGG36-9 mature sequence in uppercase letters) is provided below:

(SEQ ID NO: 762) GtgagaagcaaaaaattgtggatcgtcgcgtcgaccgcactactcatttctgttgcttttagttcatcgatcgcatcggctgctgaagaagcaaaagaaaaatatttaattggctttaatgagcaggaagctgtcagtgagtttgtagaacaagtagaggcaaatgacgaggtcgccattctctctgaggaagaggaagtcgaaattgaattgcttcatgaatttgaaacgattcctgttttatccgttgagttaagcccagaagatgtggacgcgcttgaactcgatccagcgatttcttatattgaagaggatgcagaagtaacgacaatgGCGCAATCAGTGCCATGGGGAATTAGCCGTGTGCAAGCCCCGGCTGCCCATAACCGTGGATTGACAGGTTCTGGTGTAAAAGTTGCTGTCCTCGATACAGGTATTTCCACTCATCCAGACTTAAATATTCGTGGTGGCGCTAGCTTTGTACCAGGGGAACCATCCACTCAAGATGGGAATGGGCATGGCACGCATGTGGCCGGGACGATTGCTGCTCTAAACAATTCGATTGGCGTACTTGGCGTAGCGCCGAGCGCGGAACTATACGCTGTTAAAGTATTAGGGGCGAGCGGTGGGGGCGCCATCAGCTCGATTGCCCAAGGATTGGAATGGGCAGGGAACAATGGCATGCACGTTGCTAATTTGAGTTTAGGAAGCCCTTCGCCAAGTGCCACACTTGAGCAAGCTGTTAATAGCGCGACTTCTAGGGGCGTTCTTGTTGTAGCGGCATCTGGAAATTCGGGTGCAGACTCAATCAGCTATCCGGCCCGTTATGCGAACGCAATGGCAGTCGGAGCTACTGACCAAAACAACAACCGCGCCAGCTTTTCACAGTATGGCGCAGGGCTTGACATCGTCGCACCAGGTGTAAACGTGCAGAGCACATACCCAGGTTCAACGTATGCCAGCTTAAACGGTACATCGATGGCTACTCCTCATGTTGCAGGTGCAGCAGTCCTTGTTAAACATAAGAACCCATCTTGGTCCAATGTACGAATCCGCGATCATCTAAAGAAAACGGCAACGAGCTTAGGAAGCACGAACTTGTATGGAAGCGGACTTGTCAATGCCGAAGCTGCAACTCGTTAA

The protein sequence of the GG36-9 variant (the signal sequence is shownin lower case letters, propeptide in lower case, underlined text, andGG36-9 mature protease sequence in uppercase letters) is provided below:

(SEQ ID NO: 763) vrskklwivastallisvafsssiasaaeeakekvligfneqeavsefveqveandevailseeeeveiellhefetipvlsvelspedvdaleldpaisyieedaevttmAQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGGGAISSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGADSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAVLVKHKNPSWSNVRIRDHLKKTATSLGSTNLYGSGLVNAEAATR

To create the NHJ3 variants using the QCLSD kit, mutagenic primers weredesigned for each of the variants according to the manufacturer'sprotocol. The mutagenesis reaction for each variant consisted of 0.5 μlof 10× Buffer, 0.5 μL of pRA68 plasmid DNA (168 ng/μL), 0.5 μl forwardmutagenic primer (25 μM), 0.5 μl reverse mutagenesis primer (25 μM), 1μl dNTPs (supplied in the QCLSD kit), 1.5 ul Quik solution (supplied inthe QCLMS kit), 1 μl Enzyme blend (supplied in the QCLSD kit), and 40 μlof distilled, deionized water to make up a 50 μL reaction volume as perthe manufacturer's instructions. The cycling program was 1 cycle at 95°C. for 2 minutes, 18 cycles of 95° C. for 20 seconds, 60° C. for 10seconds and 68° C. for 3 minutes, 22 seconds, and a final cycle of 68°C. for 5 minutes. Next, 1 μL of DpnI restriction enzyme supplied in thekit was used to digest the plasmid DNA in the reaction, and then 2 μL ofthe reaction was used to transform TOP 10 E. coli competent cells(Invitrogen). The E. coli transformants were selected on Luria brothmedium plates containing 50 μg/mL(ppm) carbenicillin after overnightgrowth at 37° C. Plasmid DNA was extracted from 4-8 E. coli coloniesgrown in LA medium containing 50 μg/mL(ppm) carbenicillin using theQIAprep spin miniprep kit (Qiagen). The plasmids were sequenced toconfirm the presence of the desired mutations. The variant plasmids werethen transformed into B. subtilis cells as described in Example 20. TheB. subtilis variant strains were grown up as described in Example 20 forfurther biochemical analysis, such as protein content determinationusing the eglin c inhibition assay (Example 19) and a BMI microswatchcleaning assay (Test Methods 4-6, Example 19). The results are providedbelow in Tables 23-1 and 23-2. The PIs are relative to GG36. In thefollowing Tables, the detergent compositions (“Det.”) correspond tothose shown in Table 19-4, above. Also, as indicated, the amino acidposition is listed according to BPN′ numbering.

TABLE 23-1 NHJ3 Multiple Mutation Variants with BMI Cleaning PerformanceIndices of at Least 1.1 Relative to GG36 in Detergents 7, 8, or 9 at 16°C. Variant Variant Sequence Relative to GG36 (Using BPN′ Numbering) GG36NHJ3-1 S103A-V104I-G159D-A232V-Q236H-Q245R-N248D-N252K NHJ3-2S101G-V104I-G159D-A232V-Q236H-Q245R-N248D-N252K NHJ3-3S101G-S103A-G159D-A232V-Q236H-Q245R-N248D-N252K NHJ3-4S101G-S103A-V104L-A232V-Q236H-Q245R-N248D-N252K NHJ3-5S101G-S103A-V104L-G159D-Q236H-Q245R-N248D-N252K NHJ3-6S101G-S103A-V104L-G159D-A232V-Q245R-N248D-N252K NHJ3-7S101G-S103A-V104L-G159D-A232V-Q236H-N248D-N252K NHJ3-8S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N252K NHJ3-9S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N248D

TABLE 23-2 NHJ3 Multiple Mutation Variants with BMI Cleaning PerformanceIndices of at Least 0.3 Relative to GG36 in Detergents 10 or 11 at 16°C. Variant Variant Sequence Relative to GG36 (Using BPN′ Numbering) GG36NHJ3-1 S103A-V104I-G159D-A232V-Q236H-Q245R-N248D-N252K NHJ3-2S101G-V104I-G159D-A232V-Q236H-Q245R-N248D-N252K NHJ3-3S101G-S103A-G159D-A232V-Q236H-Q245R-N248D-N252K NHJ3-4S101G-S103A-V104L-A232V-Q236H-Q245R-N248D-N252K NHJ3-5S101G-S103A-V104L-G159D-Q236H-Q245R-N248D-N252K NHJ3-6S101G-S103A-V104L-G159D-A232V-Q245R-N248D-N252K NHJ3-7S101G-S103A-V104L-G159D-A232V-Q236H-N248D-N252K NHJ3-8S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N252K NHJ3-9S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N248D

Example 24 Construction and Cleaning Performance of NHJ5 Set of GG36Variants

The NHJ5 set of variants described herein are based on a variant of GG36(referred to as GG36-7) containing the following mutations: S101G,S103A, V104I, G159D, A232V, Q245R, N248D, and (BPN′ numbering). Thesevariants were created using the QUIKCHANGE® Lightning MultiSite-Directed Mutagenesis Kit (“QCLMS kit”) with the pRA96 plasmid asthe DNA template (see FIG. 8). The mutations incorporated included:H243R (H249R), E265F (E271F), D157E (D159E), A156E (A158E), A156E-D157G(A158E-D159E), T22A, N60E (N62E), N232R (N238R), D242R (D248R), T247R(T253R), S24R, N74D (N76D) {GG36 Numbering and BPN′ Numbering Shown inParentheses.

The variants were generated using the methods described in Example 23.The B. subtilis variant strains were grown up as described in Example 20for further biochemical analysis, such as protein content determinationusing the eglin c inhibition assay (Example 19) and the BMI microswatchcleaning assay (Example 19). The results are provided below in Table24-1. The PI values are relative to GG36. In the following Tables, thedetergent compositions (“Det.”) correspond to those shown in Table 19-4,above. Also, as indicated, the amino acid position is listed accordingto BPN′ numbering.

The DNA sequence of GG36-7 variant (the signal sequence is shown inlower case letters, propeptide in lower case, underlined text, andGG36-7 mature protease sequence in uppercase letters) is provided below:

(SEQ ID NO: 764) gtgagaagcaaaaaattgtggatcgtcgcgtcgaccgcactactcatttctgttgcttttagttcatcgatcgcatcggctgctgaagaagcaaaagaaaaatatttaattggctttaatgagcaggaagctgtcagtgagtttgtagaacaagtagaggcaaatgacgaggtcgccattctctctgaggaagaggaagtcgaaattgaattgcttcatgaatttgaaacgattcctgttttatccgttgagttaagcccagaagatgtggacgcgcttgaactcgatccagcgatttcttatattgaagaggatgcagaagtaacgacaatgGCGCAATCAGTGCCATGGGGAATTAGCCGTGTGCAAGCCCCGGCTGCCCATAACCGTGGATTGACAGGTTCTGGTGTAAAAGTTGCTGTCCTCGATACAGGTATTTCCACTCATCCAGACTTAAATATTCGTGGTGGCGCTAGCTTTGTACCAGGGGAACCATCCACTCAAGATGGGAATGGGCATGGCACGCATGTGGCCGGGACGATTGCTGCTCTAAACAATTCGATTGGCGTACTTGGCGTAGCGCCGAGCGCGGAACTATACGCTGTTAAAGTATTAGGGGCGAGCGGTGGGGGCGCCATCAGCTCGATTGCCCAAGGATTGGAATGGGCAGGGAACAATGGCATGCACGTTGCTAATTTGAGTTTAGGAAGCCCTTCGCCAAGTGCCACACTTGAGCAAGCTGTTAATAGCGCGACTTCTAGGGGCGTTCTTGTTGTAGCGGCATCTGGAAATTCGGGTGCAGACTCAATCAGCTATCCGGCCCGTTATGCGAACGCAATGGCAGTCGGAGCTACTGACCAAAACAACAACCGCGCCAGCTTTTCACAGTATGGCGCAGGGCTTGACATCGTCGCACCAGGTGTAAACGTGCAGAGCACATACCCAGGTTCAACGTATGCCAGCTTAAACGGTACATCGATGGCTACTCCTCATGTTGCAGGTGCAGCAGTCCTTGTTAAACAAAAGAACCCATCTTGGTCCAATGTACGAATCCGCGATCATCTAAAGAATACGGCAACGAGCTTAGGAAGCACGAACTTGTATGGAAGCGGACTTGTCAATGCCGAAGCTGCAACTCGT

The protein sequence of GG36-7 variant (signal sequence is shown inlower case letters, propeptide in lower case, underlined text, andGG36-7 mature protease sequence in uppercase letters) is provided below:

(SEQ ID NO: 765) vrskklwivastallisvafsssiasaaeeakekvligfneqeavsefveqveandevailseeeeveiellhefetipvlsvelspedvdaleldpaisyieedaevttmAQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGGGAISSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGADSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAVLVKQKNPSWSNVRIRDHLKNTATSLGSTNLYGSGLVNAEAATR

TABLE 24-1 NHJ5 Multiple Mutation Variants with BMI Cleaning PerformanceIndices of at Least 0.6 Relative to GG36 in Detergents 7-11 at 16° C.Variant Variant Sequence Relative to GG36 (BPN′ Numbering) GG36 GG36-7S101G-S103A-V104I-G159D-A232V-Q245R-N248D NHJ5-1S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F NHJ5-2S101G-S103A-V104I-G159D-A232V-Q245R-N248D-N238R NHJ5-3S101G-S103A-V104I-G159D-A232V-Q245R-N248D-N248R NHJ5-4S101G-S103A-V104I-G159D-A232V-Q245R-N248D-T253R NHJ5-5S101G-S103A-V104I-G159D-A232V-Q245R-N248D-S24R NHJ5-6S101G-S103A-V104I-G159D-A232V-Q245R-N248D-N76D NHJ5-7S101G-S103A-V104I-G159E-A232V-Q245R-N248D-H249R NHJ5-8S101G-S103A-V104I-G159E-A232V-Q245R-N248D-E271F NHJ5-9S101G-S103A-V104I-A158E-A232V-Q245R-N248D-H249R NHJ5-10S101G-S103A-V104I-A158E-A232V-Q245R-N248D-E271F NHJ5-11T22A-S101G-S103A-V104I-G159D-A232V-Q245R-N248D- H249R NHJ5-12T22A-S101G-S103A-V104I-G159D-A232V-Q245R-N248D- E271F NHJ5-13N62E-S101G-S103A-V104I-G159D-A232V-Q245R-N248D- H249R NHJ5-14N62E-S101G-S103A-V104I-G159D-A232V-Q245R-N248D- E271F

Example 25 Construction of NHJ2 Combinatorial Library

This Example describes the construction of a GG36 combinatorial libraryinvolving one or more of the following mutations: A16S, T22A, S101A,S103G, V104L, L111V, S128N, and L148I (BPN′ numbering). The pHPLT-GG36B. subtilis expression plasmid was provided to DNA 2.0 Inc. for thegeneration of NHJ2 combinatorial library. A ligation reaction of theconstructed NHJ2 library was provided by DNA 2.0, Inc. fortransformation in the B. subtilis strain (genotype: ΔaprE, ΔnprE,amyE::xylRPxylAcomK-phleo). The variants generated containing one orseveral of the mutations described herein are tested for cold watercleaning applications using methods and detergent compositions describedherein.

Example 26 Construction of Additional Libraries and GG36 Variants

Additional libraries and variants are constructed using the followingset of mutations: A1R, A230E, E271L, G115R, G20R, H249R, K235F,K27V/F/L, L75E, L82R, N18R, N269R, N43D, N43R, N76D, R45T, S212F, S242R,S24R, S78R, S9A, T22R, V121E, V244R, V28E, V30E, V4R, W241R (BPN′numbering). The variants generated containing one or more of thesemutations are tested for cold water cleaning applications using methodsand detergent compositions described herein.

Additional sets of GG36 variants are constructed and tested for coldwater cleaning applications using methods and detergent compositionsdescribed herein include: G20R-N43R-H249R, G20R-T22R-N43R,G20R-N43R-S242R, G20R-N43R-E271L, G20R-N43R-V244R, G20R-S24R-N43R-S242R,S9A-T22R-S78R-S212F-W241R, S9A-G20R-N43R-S212F, S9A-N43R-S212F,G20R-N43R-S212F, G20R-T22R-N43R-S212F, S24R-S78R-S212F, S9A-N43R-S78R,S9A-N43R-S78R-S242R, S9A-G20R-N43R-S78R, G20R-S24R-N43R-S78R-S242R,T22R-S24R-S78R-S212F, S9A-G20R-N43R-S78R-S242R, G20R-N43R-S78R-H249R,G20R-N43R-S78R, S9A-S78R-S212F, S9A-T22R-N43R-S78R, S9A-G20R-S24R-N43R,S9A-T22R-S78R-S212F, V4R-S9A-T22R-S78R-S212F, G20R-S24R-N43R,A1R-S9A-N43R, G20R-S24R-N43R-G115R, S9A-S24R-N43R, G20R-T22R-S24R-N43R,A1R-S24R-N43R, S9A-G20R-S24R-N43R-S242R, S9A-G20R-T22R-S78R-S212F,S9A-S24R-N43R-V244R, S9A-S24R-N43R-S242R, V4R-S9A-T22R-S24R-S212F andT22R-S24R-N43R (BPN′ numbering).

Example 27 Construction and Cleaning Performance of the GG36 LibraryWCE2

The WCE2 combinatorial library was generated by DNA 2.0, Inc. using thepHPLT-GG36 B. subtilis expression plasmid. A ligation reaction of theconstructed WCE2 library was provided by DNA 2.0, Inc. fortransformation in the B. subtilis strain (genotype: ΔaprE, ΔnprE,amyE::xylRPxylAcomK-phleo). The set of mutations used to generate theWCE2 library are A230E, G20R, H249R, N18R, N43R/D, N76D, R45T, S242R,and S24R (BPN′ numbering). The variants generated containing one or moreof these mutations are tested for cold water cleaning applications usingmethods and detergent compositions described herein.

Example 28 Construction and Cleaning Performance of the WCE3 Set of GG36Variants

This Example describes the WCE3 set of mutants based on the GG36variants, GG36-7 (Example 23) and GG36-9 (Example 22). These variantsare: S101G-S103A-V104I-A232V-Q245R-N248D,S101G-S103A-V104I-G159D-A232V-Q245R,S101G-S103A-V104I-G159R-A232V-Q245R-N248D,S101G-S103A-V104I-G159D-A232V-Q245R-N248R,S101G-S103A-V104I-A232V-Q245R, S101G-S103A-V104I-A232V-Q245R-N248R,S101G-S103A-V104I-G159R-A232V-Q245R-N248R, and S101G, S103A, V104I,A232V, Q236H, Q245R, and N252K. They were created using the QuikChange®Lightning Multi Site-Directed Mutagenesis Kit (QCLMS kit; Stratagene)with the pRA96 plasmid as the DNA template described in Example 23. Thevariants generated will be tested for cold water cleaning applicationsusing methods and detergent compositions described in this application.

Example 29 Construction of Additional Libraries and Variants of GG36

This Example describes the construction of GG36 variants and librariesusing one or more of the following mutations: A16S, T22A, S24R, N62E,N76D, E89P, S101A/G, S103G/A, V104L/I, L111V, S128N, P129E, A232V,L148I, A158E, G159D/E, S166D, R186H, S188D, Y209E, Q236H, N238R, Q245R,N248D/R, H249R, N252K/R, T253R, E271F (BPN′ numbering) using a B.subtilis expression plasmid (e.g., pHPLT-GG36; FIG. 6). The variantsgenerated containing one or more of these mutations are tested for coldwater cleaning applications using methods and detergent compositionsdescribed herein.

Example 30 Construction of Additional Libraries and Variants of GG36

This Example describes the construction of GG36 variants and librariesin B. subtilis using one or more of the following mutations (BPN′numbering): A1R, Q2S, Q2M, Q2A, Q2R, Q2W, S3R, V4R, V4S, V4C, I8A, S9A,S9F, S9W, R10S, R10A, R10H, R10M, Q12F, Q12R, P14K, P14F, P14Q, A15R,A15F, A16S, H17R, H17M, H17F, N18R, N18K, G20F, G20K, G20R, T22A, T22R,T22Y, T22V, T22Q, T22L, T22W, G23A, G23S, G23F, S24R, S24F, S24W, S24Q,S24H, S24L, G25V, G25F, G25R, V26F, K27L, K27F, K27R, K27V, V28A, V28N,V28E, A29T, V30E, L31F, T33S, T33G, T33D, G34P, I35M, S36T, S36F, S36R,T38L, T38F, T38R, P40N, P40L, P40T, P40W, P40H, P40R, L42I, N43A, N43F,N43I, N43S, N43R, N43M, N43W, N43D, R45T, G46R, A48R, F50C, V51W, V51F,V51H, P52F, P52E, P52N, P55Y, T57R, Q59A, Q59F, Q59R, D60P, D60Q, D60A,N62E, N62Q, G63V, G63M, G63T, G63I, G63A, G63S, G63H, G63Q, G63D, G63E,G63P, H64F, H64T, V68A, V68C, A69N, A69T, A69P, A69W, T71G, I72C, A74C,L75A, L75F, L75E, L75R, N76D, S78R, S78N, S78I, I79W, I79Q, V81R, L82F,L82T, L82V, L82R, L82M, A85M, P86W, P86L, P86I, E89P, E89T, E89G, E89H,E89L, E89V, E89W, E89F, E89I, Y91N, Y91F, A92F, K94N, S99F, S99T, S99P,S99G, S99M, G100S, G100N, G100Q, G100I, S101A, S101N, S101G, S101T,S101D, S101E, S101P, S101F, G102A, G102T, G102N, G102H, G102E, S103G,S103N, S103D, S103A, V104L, V104I, V104E, V104D, S105T, S105E, S105Q,S106G, S106T, S106E, S106D, S106A, S106V, S106F, I107M, I107F, A108I,A108G, Q109M, L111V, L111I, E112V, E112L, E112Q, A114G, G115K, G115R,N116K, N116A, N116L, N117F, G118R, G118I, M119C, H120A, H120F, H120R,V121F, V121E, N123G, N123E, L124S, S128D, S128F, S128L, S128N, S128H,S128M, S128I, S128Q, P129E, S132A, S132E, A138G, S144R, V147L, L148I,A158E, G159D, G159E, G159C, S160D, S166D, S166E, Y167W, M175V, V177C,D181A, Q182R, N183I, N183D, N183M, N183F, N183R, N185E, N185V, N185I,R186H, R186K, S188E, S188D, S188R, Y192H, Y192W, A194E, A194V, A194F,D197F, I198L, I198F, V203E, V203C, T208S, Y209S, Y209N, Y209F, Y209T,Y209E, Y209H, Y209G, Y209L, P210R, P210V, P210L, G211Q, G211R, S212I,S212M, S212F, T213A, Y214F, A215N, A215D, A215E, A215H, A215F, S216F,S216A, L217E, L217N, L217D, N218D, N218P, N218E, T224A, T224G, V227I,A230E, A231I, A231C, A232V, L233C, V234F, K235F, Q236F, Q236N, Q236H,N238R, N238K, N238L, P239K, P239G, P239R, P239H, P239T, P239N, P239S,P239F, S240R, W241R, S242L, S242R, N243F, N243R, V244R, Q245R, I246S,N248D, N248V, N248I, N248R, H249R, H249T, L250I, K251R, K251S, N252I,N252F, N252R, N252K, N252H, T253I, T253R, T253F, A254C, S256N, G258R,T260V, T260I, L262D, L262H, Y263F, S265F, L267V, L267N, L267M, N269I,N269R, A270C, E271I, E271V, E271H, E271M, E271L, E271P, E271A, E271F,E271T, A272F, A272F, A272R, A273F, A273I, and T274G. The variantsgenerated containing one or more of these mutations are tested for coldwater cleaning applications using methods and detergent compositionsdescribed herein.

Example 31 Cleaning Performance Assay on BMI swatch at 60F (Test Method2)

The stain removal performance of the protease variants was determined incommercially available detergents (unless otherwise stated). Heatinactivation of commercial detergent formulas serves to destroy theenzymatic activity of any protein components while retaining theproperties of non-enzymatic components. Thus this method was suitablefor preparing commercially purchased detergents for use in testing theenzyme variants of the present invention.

Microswatches:

Microswatches of ¼″ circular diameter were ordered and delivered by CFT(Vlaardingen, The Netherlands). Single microswatches or twomicroswatches were placed vertically in each well of a 96-well MTP toexpose the whole surface area (i.e., not flat on the bottom of thewell).

BMI Microswatch Assay

Microswatches containing blood milk and ink (BMI) of 0.25 inch circulardiameter were obtained from CFT. Before cutting of the swatches, thefabric (EMPA 116) was washed with water. One microswatch was verticallyplaced in each well of a 96-well microtiter plate (hereinafter referredto as MTP) in order to expose the whole surface area (i.e., not flat onthe bottom of the well). The desired detergent solution was prepared asdescribed herein. After equilibrating the Thermomixer at 25° C., 190 μlof detergent solution was added to each well of the MTP, containingmicroswatches. To this mixture, 10 μl of the diluted enzyme solution wasadded so that the final enzyme concentration was 1 μg/ml (determinedfrom TCA assay). The MTP was sealed with tape and placed in theincubator for 30 minutes, with agitation at 1400 rpm. Followingincubation under the appropriate conditions, 100 μl of the solution fromeach well was transferred into a fresh MTP. The new MTP containing 100μl of solution/well was read at 405 nm using a MTP SpectraMax reader.Blank controls, as well as a control containing two microswatches anddetergent but no enzyme were also included.

“Pre-Washed” Swatch

This type of microswatch was pre-washed in deionised water for 20minutes at ambient temperature. After the pre-washing step, the swatcheswere put on top of paper towels to dry. The air-dried swatches were thenpunched using a ¼″ circular die on an expulsion press. Finally twomicroswatches were put into each well of a 96-well MTP vertically toexpose the whole surface area (i.e. not flat on the bottom of the well).

Detergents

For North American (NA) and Western European (WE) heavy duty liquidlaundry (HDL) detergents, heat inactivation was performed by placingpre-weighed liquid detergent (in a glass bottle) in a water bath at 95°C. for 2 hours. All detergents were purchased from local supermarketstores. Both un-heated and heated detergents were assayed within 5minutes of dissolving the detergent to accurately determine percentagedeactivated. Enzyme activity was tested by the AAPF assay.

For testing of enzyme activity in heat-inactivated detergents, workingsolutions of detergents were made from the heat inactivated stocks.Appropriate amounts of water hardness (for instance 6 gpg for NA HDL and12 gpg for WE HDL) and buffer were added to the detergent solutions tomatch the desired conditions as specified in Table 31-1 below. Thesolutions were mixed by vortexing or inverting the bottles.

TABLE 31-1 Laundry and Dish Washing Conditions Region Form DoseDetergent* Buffer gpg pH T (° C.) Laundry (heavy duty liquid andgranular) NA HDL 0.78 g/l   P&G TIDE ® 2X 5 mM HEPES 6 8.0 20 WE HDL 5.0g/L Henkel Persil 5 mM HEPES 12 8.2 40 JPN HDG 0.7 g/L P&G TIDE ® 2 mMNa₂CO₃ 6 10.0 20 *Abbreviations: Procter & Gamble (P&G);Enzymes and Equipment

Samples of reference serine proteases variants thereof were obtainedfrom filtered culture broth of cultures grown in MTP plates. Theequipment used was a Biomek FX Robot (Beckman Coulter), a SpectraMAX MTPReader (type 340; Molecular Devices), an iEMS incubator/shaker(Thermo/Labsystems); F-bottom MTPs (Costar type 9017 used for readingreaction plates after incubation); and V-bottom MTPs (Greiner 651101used for pre-dilution of supernatant). In this assay, the proteaseshydrolyze the substrate and liberate pigment and insoluble particlesfrom the substrate. Thus the rate of turbidity is a measure of enzymeactivity.

The stain removal performance of reference serine proteases and variantstherefrom on microswatches was determined on a MTP scale in commerciallyavailable heat-inactivated detergent. The reagents used were: 5 mMHEPES, pH 8.0 or 5 mM MOPS, pH 7 buffer, 3:1 Ca:Mg for medium waterhardness. (CaCl₂:MgCl₂.6H₂O); 15000 grains per gallon (gpg) stockdiluted to 6 gpg, 2 BMI (blood/milk/ink) swatches per plate: EMPA-116BMI cotton swatches processed by CFT: pre-rinsed and punched 2 swatchesper well, and heat inactivated TIDE® 2× Cold off-the-shelf detergent inwhich lack of protease activity was confirmed.

TABLE 31-2 Working Detergent Solutions Temp Detergent Detergent (C.) g/LpH Buffer gpg Protease TIDE ® 2X 16 0.98 8 5 mM HEPES 6 BPN′ Cold TIDE ®2X 32 0.98 8 5 mM HEPES 6 BPN′ Cold TIDE ® 2X 16 0.98 7 5 mM MOPS 6 BPN′Cold

The incubator was set at the desired temperature (16° C. or 32° C.). 10μL samples from the master dilution plate of ˜10 ppm enzyme was added toBMI 2-swatch plates with 190 μL working detergent solutions listedabove. The volume was adjusted to give final concentration of 0.5 ppmfor variants in the assay plates. The plates were immediatelytransferred to iEMS incubators and incubated for 30 minutes with 1400rpm shaking at given temperature. Following incubation, 100 μL ofsupernatant was transferred into a new 96-well plate and the absorbancewas measured in MTP Reader at 405 nm and/or 600 nm. Control wells,containing 1 or 2 microswatches and detergent without the addition ofprotease samples were also included in the test. The measurement at 405nm provides a higher value and tracks pigment removal, while themeasurement at 600 nm tracks turbidity and cleaning.

Calculation of the Stain Removal Activity for All Microswatch AssayMethods:

The absorbance value obtained was corrected for the blank value(substrate without enzyme), providing a measure of hydrolytic activity.For each sample (variant) the performance index was calculated. Theperformance index compares the performance of the variant (actual value)and the standard enzyme (theoretical value) at the same proteinconcentration. In addition, the theoretical values can be calculated,using the parameters of the Langmuir equation of the standard enzyme.

Performance Index

The performance index compares the performance of the variant (actualvalue) and the standard or reference protease (theoretical value) at thesame protein concentration. In addition, the theoretical values can becalculated, using the parameters of the binding curve (i.e., Langmuirequation) of the standard protease. A performance index (PI) that isgreater than 1 (PI>1) identifies a better variant as compared to thestandard (e.g., wild-type), while a PI of 1 (PI=1) identifies a variantthat performs the same as the standard, and a PI that is less than 1(PI<1) identifies a variant that performs worse than the standard. Thus,the PI identifies winners, as well as variants that are less desirablefor use under certain circumstances.

TCA Assay for Protein Content Determination in 96-well Microtiter Plates

For BPN′ (e.g., reference protease) and BPN′ variants, this assay wasstarted using filtered culture supernatant from microtiter plates grown3-4 days at 33° C. with shaking at 230 rpm and humidified aeration. Afresh 96-well flat bottom microtiter plate (MTP) was used for the assay.First, 100 μL/well of 0.25 N HCl was placed in each well. Then, 50 μL offiltered culture broth was added. The light scattering/absorbance at 405nm (use 5 sec mixing mode in the plate reader) was then determined, inorder to provide the “blank” reading. For the test, 100 μL/well of 15%(w/v) trichloroacetic acid (TCA) was placed in the plates and incubatedbetween 5 and 30 min at room temperature. The lightscattering/absorbance at 405 nm (use 5 sec mixing mode in the platereader) was then determined.

The equipment used was a Biomek FX Robot (Beckman Coulter) and aSpectraMAX (type 340; Molecular Devices) MTP Reader; the MTP's were fromCostar (type 9017). The equipment used was a Biomek FX Robot (BeckmanCoulter) and a SpectraMAX type 340 (Molecular Devices) MTP Reader; andthe MTPs were type 9017 (Costar).

The calculations were performed by subtracting the blank (no TCA) fromthe test reading with TCA to provide a relative measure of the proteincontent in the samples. If desired, a standard curve can be created bycalibrating the TCA readings with AAPF assays of clones with knownconversion factors. However, the TCA results are linear with respect toprotein concentration from 50 micrograms to 500 micrograms protein perml (ppm) and can thus be plotted directly against enzyme performance forthe purpose of choosing good-performing variants. The turbidity/lightscatter increase in the samples correlates to the total amount ofprecipitable protein in the culture supernatant.

AAPF Protease Assay in 96-well Microtiter Plates

In order to determine the protease activity of the proteases andvariants thereof of the present invention, the hydrolysis ofN-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-p-nitroanilide(suc-AAPF-pNA) was measured. The reagent solutions used were: 100 mMTris/HCl, pH 8.6, containing 0.005% TWEEN®-80 (Tris dilution buffer);100 mM Tris buffer, pH 8.6, containing 10 mM CaCl₂ and 0.005% TWEEN®-80(Tris/Ca buffer); and 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stocksolution) (Sigma: S-7388). To prepare a suc-AAPF-pNA working solution, 1ml suc-AAPF-pNA stock solution was added to 100 ml Tris/Ca buffer andmixed well for at least 10 seconds. The assay was performed by adding 10μl of diluted protease solution to each well, immediately followed bythe addition of 190 μl 1 mg/ml suc-AAPF-pNA working solution. Thesolutions were mixed for 5 sec., and the absorbance change in kineticmode (20 readings in 5 minutes) was read at 410 nm in an MTP reader, at25° C. The protease activity was expressed as AU (activity=ΔOD·min⁻¹ml⁻¹).

Stability Assay

The stability of protease variants was determined in the presence of 40%concentrated solution of detergent composition 3 shown in Table 1-3diluted in water. The reagents used were the detergent composition 3shown in Table 1-3 diluted to 50% in Milli-Q water, 10 mM MES 0.01%Tween 80 pH 5.8 master dilution buffer, AAPF reagents: see protocol AAPFassay. The equipment used was F-bottom MTP (Corning 9017) for dilutionof diluted enzyme into detergent as well as for suc-AAPF-pNA plates,Biomek FX (Beckman Coulter), Spectramax Plus 384 MTP Reader (MolecularDevices), iEMS Incubator/Shaker (1 mm amplitude) (Thermo ElectronCorporation), sealing tape: Nunc (236366), Circulating Reservoir(Beckman Fx).

The detergent composition 3 shown in Table 1-3 was initially diluted to50% in water. This detergent was kept at room temperature and cycledthrough the circulating reservoir. The iEMS incubators/shakers(Thermo/Labsystems) were pre-set at 43°. Culture supernatants werediluted into plates containing master dilution buffer to a concentrationof ˜20 ppm (master dilution plate). 40 μl of sample from the masterdilution plate was added to plates containing 160 μl50% detergentcomposition 3 shown in Table 1-3 to give a final incubationconcentration of 4 ppm. The contents were mixed and kept at roomtemperature and triplicate AAPF assays were performed immediately onthese plates and recorded as unstressed reads. The AAPF assay wasmodified such that 20 μL of sample from the step above was added to 190μL of suc-AAPF-pNA working solution. The plates were immediately coveredwith sealing tape and placed in 43° C. iEMS shakers for 30 min at 650rpm. Following 30 minutes of incubation, triplicate AAPF assays wereperformed on these stress plates and recorded as stressed reads. Thestability of the samples was determined by calculating the ratio of theresidual and initial AAPF activity as follows: Residual Activity(%)=[mOD·min−1 stressed]*100/[mOD·min−1 unstressed].

LAS/EDTA Stability Assay

The stability of protease variants in the presence of a representativeanionic surfactant (LAS=linear alkylbene sulfonate, sodiumdodecylbenzenesulfonate-DOBS) and di-sodium EDTA was measured afterincubation under defined conditions and the residual activity wasdetermined using the AAPF assay. The reagents used were dodecyllbenzenesulfonate, sodium salt (DOBS, Sigma No. D-2525), TWEEN®-80 (Sigma No.P-8074), di-sodium EDTA (Siegfried Handel No. 164599-02), HEPES (SigmaNo. H-7523), unstress buffer: 50 mM HEPES (11.9 g/l)+0.005% TWEEN®-80,pH 8.0, Stress buffer: 50 mM HEPES (11.9 g/l), 0.1% (w/v) DOBS (1 g/l),10 mM EDTA (3.36 g/l), pH 8.0, reference protease and protease variantculture supernatants, containing 200-400 μg/ml protein. The equipmentused was V- or U-bottom MTP as dilution plates (Greiner 651101 and650161 respectively), F-bottom MTP (Corning 9017) for unstress andLAS/EDTA buffer as well as for suc-AAPF-pNA plates, Biomek FX (BeckmanCoulter), Spectramax Plus 384 MTP Reader (Molecular Devices), iEMSIncubator/Shaker (1 mm amplitude) (Thermo Electron Corporation), sealingtape: Nunc (236366).

The iEMS incubator/shaker (Thermo/Labsystems) was set at 29° C. Culturesupernatants were diluted into plates containing unstress buffer to aconcentration of ˜25 ppm (master dilution plate). 20 μl of sample fromthe master dilution plate was added to plates containing 180 unstressbuffer to give a final incubation concentration of 2.5 ppm. The contentswere mixed and kept at room temperature and a AAPF assay was performedon this plate. 20 μl of sample from the master dilution plate was alsoadded to plates containing 180 μl stress buffer (50 mM HEPES (11.9 g/l),0.1% (w/v) DOBS (1 g/l), 10 mM EDTA (3.36 g/l), pH 8.0). The solutionswere mixed and immediately placed in 29° C. iEMS shaker for 30 min at400 rpm. Following 30 minutes of incubation, a AAPF assay was performedon the stress plate. The stability of the samples was determined bycalculating the ratio of the residual and initial AAPF activity asfollows: Residual Activity (%)=[mOD·min−1 stressed]*100/ [mOD·min−1unstressed].

Performance Data of BPN′ Variants

The below tables exemplify the performance of the Series 1 BPN′ coldwater protease variants described in this patent as assayed by TestMethod 2.

Table 31-3: Performance of Single Mutation Proteases

-   Library Parent: BPN′ Wild type (Enzyme of SEQ ID NO:2)-   PI reference: calculated relative to BPN′ WT for the BMI pH8 16° C.    assay.-   Detergents: Commercial heat deactivated Tide 2× Cold    Description:

In this Example, results of experiments conducted to determine proteinexpression, stain removal activity, LAS stability, and AAPF activity(tests of properties of interest) of BPN′ and BPN′-variants aredescribed. BPN′ single variants were constructed using PCR fusion asdescribed in USPA 2009/46156, filed Jun. 3, 2009, herein incorporated byreference. The results were obtained using the methods described in TestMethod 2. As described throughout, functionality of the BPN′ variantswas quantified as a performance index (PI), which is the ratio ofperformance of a variant to a parent protein. Table 32-1 providesPerformance index values (PI) of subtilisin BPN′ variants. Performanceindices less than or equal to 0.1 were fixed to 0.1 and indicated inbold italics in the table. Also, for the stability measure, if thePerformance index of activity in the stability assays was less than orequal to 0.1, the associated stability performance index was fixed to0.1.

TABLE 31-3 Performance Index Values for BPN′ Variants PI BMI PI BMI PIBMI LAS- specific BPN′ TCA pH 8 pH 7 pH 8 EDTA AAPF Position variant PI16 C 16 C 32 C PI PI 2 Q002W 0.2 3.8 6.9 2.4

0.5 5 P005K 0.2 4.5 2.6 0.7 0.6 0.1 5 P005L 0.2 1.5 1.4 1.5

0.5 5 P005Y 0.2 3.3 3.3 2.2

0.4 7 G007T 0.2 3.0 2.4 3.0 0.1 0.2 8 V008G 0.3 1.4 0.5 1.4

0.4 8 V008K 0.2 3.1 3.0 3.1 0.2 0.5 8 V008P 0.2 2.7 3.0 2.9

0.6 11 I011G 0.2 2.2 2.2 1.6 0.4 0.3 11 I011H 0.2 36.5 114.2  1.9 0.80.4 11 I011S 0.3 1.3 1.7 1.3 0.3 1.0 12 K012L 0.2 3.1 4.6 2.0 0.6 0.3 13A013M 0.2 1.4

0.8

16 L016W 0.2 3.4 6.2 3.2 0.1 0.4 23 G023S 0.2 10.8 4.1 2.6 1.1 0.1 26V026H 0.5 1.5 0.9 1.2 1.1 0.9 26 V026W 0.3 2.3 1.2 2.1 1.1 1.0 26 V026Y0.3 1.3 1.3 1.5 1.0 1.0 27 K027P 0.2 1.3 3.4 2.1 1.1 0.5 28 V028Q 0.22.8 2.0 1.4 1.6 0.3 28 V028S 0.2 5.7 5.2 3.4 1.1 0.4 28 V028T 0.6 1.31.2 1.1 1.0 1.2 29 A029C 0.8 1.3 1.2 1.1 0.9 0.8 29 A029D 0.2 3.0 2.72.3 0.9 0.4 29 A029S 0.5 1.6 1.1 1.2 1.1 1.0 29 A029T 0.2 2.0 1.6 1.61.1 0.5 29 A029V 0.4 1.9 1.5 1.6 1.0 0.8 30 V030D 0.2 2.3 2.3 2.0 0.90.2 30 V030E 0.2 30.2 33.3  4.3 0.8 0.2 30 V030G 0.2 7.6 3.7 1.4

30 V030T 0.3 1.4 1.3 1.4 0.9 0.4 31 I031E 0.2 2.6 2.0 2.4 1.1 1.1 31I031G 0.2 5.4 3.7 2.4 1.1 0.4 31 I031H 0.3 2.0 1.6 1.8 1.3 1.2 31 I031K0.3 1.8 1.3 1.4 1.2 1.0 31 I031N 0.2 2.3 2.0 2.0 1.3 0.7 31 I031Q 0.25.4 3.5 3.1 1.1 0.6 31 I031S 0.2 3.7 2.8 2.9 1.1 0.9 31 I031Y 0.2 4.13.0 3.1 0.9 0.9 33 S033F 0.5 1.4 0.6 1.0

33 S033H 0.5 1.7 0.8 1.2

36 D036L 0.3 1.9 1.3 1.4

0.7 37 S037P 0.5 1.5 0.9 1.1 0.4 0.7 41 D041A 0.2 2.5

0.8 0.1 0.2 41 D041C 0.3 1.7 0.3 1.1

0.5 41 D041M 0.2 10.4

0.6

41 D041N 0.3 2.1 1.3 1.4

0.5 41 D041S 0.2 3.1 1.5 1.2 0.5 0.3 42 L042S 0.2 32.8

0.4

42 L042Y 0.2 3.6 4.4 1.9

0.4 44 V044H 0.2 1.6 2.6 1.6 0.5 0.4 44 V044Q 0.2 1.5 2.9 1.5 0.6 0.3 44V044T 0.3 1.3 1.9 1.3 0.7 1.3 46 G046F 0.4 1.4 1.0 1.0 1.0 0.9 46 G046L0.3 1.6 1.4 1.4 0.8 0.8 46 G046M 0.4 1.3 0.8 1.1 0.9 1.0 46 G046V 0.24.9 3.3 2.1 0.8 0.7 47 G047T 0.2 4.5 4.8 2.3 0.5 0.1 47 G047W 0.2 2.62.4 2.2 0.5 0.2 49 S049I 0.2 2.2 2.5 2.1 0.5 0.1 49 S049V 0.2 1.6 1.42.1 0.4 0.3 50 M050D 0.2 6.4 0.9 0.9 1.0 0.2 50 M050I 0.3 1.6 0.9 1.10.9 0.6 50 M050R 0.3 1.4 0.7 1.1 1.0 0.8 51 V051A 0.4 1.4 1.3 1.2 0.90.4 51 V051G 0.2 3.4 4.3 1.4

51 V051S 0.3 1.8 2.0 1.4 0.9 0.4 52 P052C 0.3 1.4 1.6 0.9 1.0 0.4 52P052I 0.5 1.3 1.5 1.1 0.8 0.6 52 P052L 0.5 1.3 1.2 1.3 0.8 0.6 52 P052M0.4 1.4 1.3 1.1 0.7 0.5 52 P052V 0.3 1.8 1.6 1.0 1.0 0.5 52 P052W 0.41.5 1.3 1.4 1.0 0.8 52 P052Y 0.4 1.6 1.4 1.3 1.0 0.6 54 E054R 0.2 1.51.8 1.7 0.6 0.6 56 N056G 0.3 1.6 1.1 1.2 0.4 0.5 56 N056I 0.2 1.7 1.31.4 0.6 0.5 56 N056K 0.2 2.1 1.5 1.9 0.5 0.4 56 N056M 0.3 1.5 1.2 1.20.5 0.4 56 N056Q 0.3 1.7 1.3 1.2 0.8 0.4 56 N056R 0.2 4.9 4.0 2.2 0.50.3 56 N056V 0.3 1.4 1.3 1.0 0.5 0.5 56 N056Y 0.3 1.3 1.3 1.3 0.7 0.4 57P057I 0.3 2.2 2.0 1.6 0.4 0.3 57 P057K 0.2 1.5 1.8 1.1 0.3 0.3 57 P057L0.2 2.6 2.0 1.5 0.4 0.3 57 P057R 0.2 2.3 3.1 1.5 0.4 0.4 57 P057T 0.32.5 2.0 2.3 0.5 0.6 57 P057V 0.2 3.6 6.0 3.5 0.3 0.5 58 F058T 0.4 1.41.8 1.2 0.7 1.0 59 Q059P 0.3 1.9 2.0 1.5 0.8 0.4 59 Q059W 0.2 1.4 1.61.2 1.0 0.5 61 N061P 1.4 1.5 1.0 0.8 1.0 1.4 62 N062D 1.5 1.4 1.2 0.91.1 0.7 62 N062M 1.8 1.3 1.1 1.1 1.1 0.6 62 N062Q 1.5 1.3 1.2 1.1 1.20.6 63 S063C 1.6 1.3 1.0 0.7 1.3 0.8 63 S063Q 1.6 1.4 0.8 1.1 1.0 1.0 63S063T 1.0 1.3 1.0 1.0 1.0 0.9 65 G065Q 0.2 1.7 2.1 2.3 1.2 0.7 68 V068A1.0 1.8 1.1 1.3 1.0 0.1 68 V068G 0.5 1.8 1.3 1.4

68 V068M 1.2 1.3 0.9 1.2

68 V068S 1.3 1.7 1.3 1.1

69 A069C 0.4 1.7 0.9 1.2 0.7 0.5 69 A069D 0.3 2.0

69 A069F 0.2 13.7 1.3 0.5

69 A069H 0.2 39.0 2.2 0.7 0.7 0.1 69 A069M 0.2 7.2 0.7 0.6 0.5 0.1 69A069N 0.2 2.5 0.1

69 A069P 0.3 1.8

69 A069Q 0.2 5.2 0.1

69 A069R 0.4 2.0

69 A069T 0.5 1.6 1.0 1.0 1.0 0.7 71 T071D 0.2 41.1 34.6  1.5 0.2 0.1 71T071E 0.3 2.2 1.9 1.0 0.1 0.3 71 T071G 0.2 3.3 1.6 1.9

0.4 71 T071K 0.2 37.0 45.5  2.2 0.1 0.2 71 T071M 0.3 1.3 1.2 1.1

0.4 72 V072D 0.2 2.5 1.7 1.9 0.7 0.2 72 V072G 0.4 1.5 1.1 1.4 0.8 0.7 72V072K 0.2 2.8 2.1 2.8 1.7 0.3 72 V072Q 0.3 1.6 1.5 1.7 0.6 0.6 72 V072S0.4 1.4 1.4 1.3 0.8 0.9 72 V072T 0.6 1.4 1.0 1.1 0.9 1.0 73 A073E 0.51.6 1.2 1.1 0.2 0.8 73 A073I 0.2 22.5 15.7  6.1 0.1 0.3 73 A073K 0.2 3.52.5 2.6 0.1 0.3 73 A073M 0.2 1.7 1.4 1.5 0.3 0.4 73 A073Q 0.6 1.3 1.31.1 0.5 0.9 73 A073S 1.0 1.4 0.9 1.0 0.8 1.1 73 A073V 0.4 1.4 1.2 1.50.1 0.8 74 A074E 0.3 4.3

74 A074F 0.3 2.6

74 A074H 0.3 3.2

74 A074I 0.3 2.1

74 A074L 0.3 2.2

74 A074M 0.2 5.3

74 A074Q 0.3 2.9 0.1

74 A074R 0.3 1.9

74 A074V 0.3 3.6 0.1

74 A074W 0.3 2.5

74 A074Y 0.3 2.8 0.1

75 L075A 1.1 1.4 1.0 1.0

1.0 76 N076A 1.1 1.7 0.8 1.0

1.0 77 N077G 0.2 1.5 0.8 1.0

0.1 77 N077L 0.2 2.2 0.2 0.8 0.1 0.1 77 N077P 0.2 2.2

0.6 0.3 0.1 77 N077Q 0.2 2.1 0.8 1.2

0.4 77 N077R 0.2 2.5 0.1 0.9 0.1 0.2 77 N077S 0.3 1.7 1.3 1.2

0.8 77 N077T 0.2 4.2 1.3 1.5

0.3 78 S078W 1.0 1.3 0.7 1.0 0.3 1.1 80 G080H 0.2 11.3 32.2  3.1

0.3 81 V081D 0.2 1.4 0.8 0.6 0.1 0.1 81 V081F 0.9 1.3 0.9 1.1

0.8 81 V081H 0.7 1.5 1.0 1.0

0.7 81 V081N 0.4 1.3 0.9 1.2

0.7 81 V081Q 0.6 1.3 0.8 1.1

0.9 81 V081R 0.2 2.0 1.5 1.8

0.6 81 V081W 0.6 1.4 0.8 1.2

0.8 82 L082G 0.2 2.2 1.7 1.5 0.1 0.4 82 L082N 0.3 1.8 1.5 1.7

0.6 82 L082W 0.2 10.3 8.5 4.2 0.7 0.4 85 A085I 0.2 1.7 0.7 0.7

85 A085T 0.4 1.4 1.1 0.9 0.6 1.0 85 A085V 0.2 2.5 2.5 2.3 0.7 0.5 86P086A 0.5 1.3 1.1 1.3 0.7 1.1 86 P086G 0.4 1.4 0.9 1.3 0.1 0.8 86 P086M0.3 2.0 1.8 1.8 0.1 1.0 86 P086Q 0.3 1.5 1.6 2.0 0.1 0.9 86 P086R 0.23.5 3.8 2.7 0.7 0.7 86 P086T 0.3 1.7 1.8 1.9 0.2 1.1 86 P086W 0.4 1.41.5 1.3 0.4 1.4 86 P086Y 0.4 1.5 1.2 1.7 0.9 1.4 87 S087F 0.9 1.4 0.91.1 0.5 1.0 87 S087I 0.6 1.4 0.8 1.0 0.4 0.9 87 S087L 1.1 1.3 1.0 1.10.8 1.0 87 S087M 1.0 1.3 1.0 1.1 0.7 1.0 87 S087Q 0.8 1.3 0.9 1.0 0.80.9 87 S087V 0.9 1.3 1.0 1.2 0.9 1.2 87 S087W 0.9 1.4 1.0 1.3 0.5 1.1 88A088D 0.3 1.4 1.6 1.3 0.7 0.7 88 A088E 0.2 11.4 17.5  1.7 0.6 0.2 88A088K 0.2 2.4 3.0 1.7 0.8 0.7 88 A088P 0.2 2.2 3.4 2.0 0.9 0.6 88 A088Q0.2 1.9 2.0 1.2 0.5 0.5 89 S089D 1.2 1.4 1.0 1.1 0.8 1.0 89 S089Q 0.2108.1 37.2  0.2

89 S089V 0.7 1.3 1.3 1.2 0.7 1.3 90 L090D 0.2 2.4 2.1 2.2 0.6 0.5 90L090E 0.2 1.6 1.4 1.5 1.0 0.5 90 L090F 0.3 1.6 0.8 1.4 0.3 0.3 90 L090H0.2 2.0 2.2 2.3 1.0 0.9 90 L090P 0.3 1.6 1.7 1.6 0.9 0.9 90 L090S 0.21.6 1.9 1.6 0.7 0.9 90 L090T 0.2 3.9 3.3 2.5 0.8 1.0 91 Y091L 0.2 1.62.8 1.7 1.0 0.5 91 Y091Q 0.2 7.6 26.9  2.0 1.2 0.3 91 Y091T 0.2 3.7 8.12.7 0.9 0.6 92 A092C 0.3 2.6 2.0 1.7 0.6 0.3 92 A092I 0.3 1.7 1.5 1.60.7 0.3 92 A092M 0.2 2.5 2.6 2.9 0.6 0.2 92 A092N 0.2 6.4 7.3 5.5 0.70.2 92 A092P 0.3 1.6 2.3 2.1 0.9 0.5 92 A092V 0.3 1.4 1.3 1.7 0.6 0.5 93V093D 0.2 2.4 3.4 2.0 1.1 0.7 93 V093F 0.2 1.3 2.2 1.5 1.0 0.6 93 V093L0.6 1.4 1.1 1.0 0.8 1.0 93 V093T 0.3 1.5 1.7 1.4 1.1 1.0 94 K094C 0.21.3 1.4 1.1 1.0 0.2 94 K094R 0.4 1.4 1.5 1.3 0.8 0.4 94 K094S 0.2 4.23.8 2.5 1.0 0.2 95 V095I 0.4 2.4 1.2 1.3

96 L096F 0.5 2.0 0.8 1.4 0.9 0.6 96 L096H 0.2 1.9 0.2 0.4

96 L096I 0.8 1.6 0.9 1.0 1.2 0.4 96 L096M 0.9 1.5 1.1 1.2 0.9 0.5 96L096N 0.2 6.1 0.9 0.7

96 L096Q 0.2 31.7 6.8 3.9

96 L096S 0.2 3.8 1.0 1.5

96 L096T 0.4 2.3 1.3 1.4

96 L096V 1.0 1.4 0.9 1.3 1.1 0.1 96 L096W 0.2 5.0 1.6 2.2 1.0 0.3 96L096Y 0.3 2.0 0.7 1.2

97 G097A 0.9 1.6 1.0 1.3 1.0 0.8 97 G097C 1.0 1.7 1.1 1.1 1.0 0.5 97G097D 1.6 1.4 0.8 1.2 1.0 0.8 97 G097E 1.1 1.4 0.8 1.1 1.1 0.7 97 G097F0.3 1.9 1.0 1.4 0.9 0.3 97 G097L 0.8 1.4 1.1 1.2 1.1 0.7 97 G097M 0.81.3 1.0 1.2 1.1 0.9 97 G097P 0.4 2.4 1.6 1.3 1.1 0.7 97 G097Q 1.0 1.40.8 1.0 1.2 1.0 97 G097S 1.0 1.5 1.1 1.1 0.9 1.0 97 G097V 0.5 1.6 1.11.2 1.0 1.0 97 G097W 0.2 3.0 1.5 1.5 1.0 0.3 97 G097Y 0.4 1.4 0.9 1.00.9 0.3 99 D099C 0.7 1.5 0.8 1.1 1.1 0.6 99 D099E 1.0 1.4 0.8 1.0 1.00.8 99 D099I 0.4 1.6 0.6 1.1 1.0 0.6 99 D099M 0.6 1.4 0.7 1.2 0.9 0.7 99D099P 0.4 2.0 0.9 1.2 1.0 0.4 99 D099V 0.4 1.7 0.8 1.1 0.9 0.6 99 D099Y0.5 1.4 0.6 1.1 0.9 0.6 100 G100D 0.8 1.4 1.4 1.1 0.9 0.3 100 G100E 0.51.7 1.5 1.3 1.0 0.3 100 G100H 0.6 1.4 1.2 1.1 0.9 0.3 100 G100I 0.3 1.81.6 1.2

100 G100K 0.7 1.5 1.0 1.2 0.9 0.3 100 G100M 1.1 1.4 1.2 1.2 0.8 0.2 100G100Q 0.7 1.6 1.2 1.4 1.0 0.4 100 G100T 0.3 2.2 1.6 1.5 1.0 0.2 100G100V 0.3 2.0 1.6 1.4 1.0 0.1 100 G100Y 0.5 1.6 1.3 1.1 1.0 0.2 101S101A 1.3 1.4 1.0 1.1 1.1 1.1 101 S101E 1.4 1.5 1.2 1.1 1.1 0.7 101S101G 0.4 1.3 0.5 0.7 1.1 0.2 101 S101N 1.2 1.5 1.0 1.1 1.1 1.0 101S101P 0.3 3.6 1.4 1.8 1.1 0.3 101 S101Q 0.6 1.6 0.8 1.1 1.0 1.2 101S101T 0.9 1.3 0.7 1.2 1.0 1.1 101 S101V 0.9 1.4 0.7 1.2 0.9 1.2 102G102A 1.1 1.5 1.3 1.2 1.0 0.3 102 G102S 0.8 1.6 1.1 1.0 1.1 0.1 103Q103E 1.2 1.4 1.2 1.1 1.0 0.6 103 Q103G 0.4 1.5 1.1 1.3 1.1 0.4 103Q103H 0.9 1.4 0.9 0.9 0.9 0.7 103 Q103K 0.2 2.6 1.4 1.3 1.0 0.5 103Q103N 1.4 1.4 1.2 0.9 1.0 0.7 104 Y104L 0.2 1.5 1.5 1.2

104 Y104M 0.3 1.5 1.5 1.3 1.2 0.1 104 Y104N 0.5 1.4 1.8 1.0 1.1 0.1 104Y104T 0.4 1.3 1.7 1.4 2.0 0.1 104 Y104V 0.3 1.7 1.2 1.4

105 S105D 0.4 1.3 1.0 1.2 1.1 0.9 105 S105I 0.2 1.6 1.1 0.7 1.0 0.3 105S105R 0.2 3.0 3.4 1.0 1.2 0.2 105 S105V 0.2 2.8 3.1 2.8 1.0 0.8 106W106A 0.4 1.4 1.2 1.1 0.9 0.4 106 W106C 0.4 1.4 1.1 1.0 0.9 0.2 106W106E 0.4 1.7 1.3 1.2 1.1 0.4 106 W106F 0.8 1.3 0.8 1.3 1.0 0.4 106W106G 0.2 1.4 1.3 1.2 0.9 0.1 106 W106I 0.3 1.5 1.1 1.3 1.1 0.4 106W106L 0.3 1.6 1.1 1.4 1.1 0.3 106 W106M 0.4 1.4 1.1 1.1 0.9 0.3 106W106S 0.4 1.4 0.9 1.2 2.0 0.2 106 W106T 0.4 1.4 1.2 1.3 0.9 0.3 106W106V 0.3 1.5 1.2 1.2 0.9 0.5 107 I107R 0.2 2.3 2.3 1.9 1.2 0.1 107I107S 0.5 1.4 0.9 1.2 1.0 0.1 107 I107T 0.7 1.4 2.3 0.9 1.0 0.1 108I108S 0.2 2.4 8.6 1.3 0.9 0.1 108 I108T 0.3 1.8 1.7 1.5 0.9 0.8 110G110S 0.3 2.2 1.4 1.7 0.9 0.5 110 G110T 0.2 4.6 4.0 2.8 0.9 0.5 111I111A 0.2 3.8 2.4 1.9 0.9 0.3 111 I111C 0.4 1.9 1.5 1.5 0.9 0.9 111I111F 0.5 1.7 1.1 1.2

111 I111L 1.0 1.3 1.0 1.1 1.0 0.7 111 I111M 0.8 1.4 1.0 1.0 1.1 0.7 111I111T 0.3 3.1 1.6 1.7 1.0 0.9 111 I111V 0.7 1.6 1.0 1.3 1.0 0.8 112E112I 0.2 7.2 2.7 2.7 1.1 0.6 112 E112L 0.2 19.7 11.6  3.8 1.1 0.6 112E112T 0.3 2.9 2.1 2.0 1.0 0.9 113 W113H 0.2 1.5 1.7 1.5 1.1 0.6 114A114I 0.2 3.4 2.6 2.5 0.9 0.4 114 A114V 0.3 1.6 2.2 1.6 0.9 1.0 115I115A 0.4 1.6 1.0 1.1 0.9 0.8 115 I115E 0.3 2.3 1.7 1.6 0.9 0.7 115I115F 0.3 2.0 1.2 1.4 0.9 0.6 115 I115H 0.2 4.9 2.5 2.4 0.9 0.5 115I115M 0.9 1.5 1.0 1.1 1.0 0.9 115 I115N 0.2 8.3 8.4 2.6 1.0 0.4 115I115P 0.2 1.8 0.1 0.2

115 I115Q 0.6 1.3 0.8 1.2 1.0 1.0 115 I115R 0.4 1.4 1.0 1.2 1.0 0.9 115I115S 0.2 4.2 2.4 2.2 0.9 0.7 115 I115T 0.6 1.5 0.9 1.1 0.9 0.9 115I115V 0.8 1.4 1.0 1.3 0.9 1.6 115 I115Y 0.2 4.8 4.1 2.5 0.9 0.8 117N117K 0.2 1.9 1.9 1.8 1.0 0.9 117 N117V 0.2 5.4 4.1 2.1 1.0 0.5 117N117W 0.3 2.1 1.8 1.6 1.0 1.1 118 N118I 0.2 4.9 5.9 2.0 1.1 0.6 118N118L 0.2 1.5 1.9 1.3 1.0 1.1 118 N118V 0.2 1.4 1.9 1.5 1.0 1.0 119M119F 0.3 1.6 1.9 1.6 1.3 0.8 119 M119N 0.2 2.0 2.6 1.8 1.1 0.8 120D120Y 0.2 1.3 1.7 1.3 1.0 0.7 122 I122R 0.2 2.8

122 I122S 0.2 6.5 6.1 2.6 1.9 0.4 122 I122T 0.3 1.8 1.8 1.1 1.4 0.9 123N123A 0.8 1.7 1.4 1.1

123 N123C 1.0 1.5 1.4 1.4 0.5 0.1 123 N123G 0.5 1.8 1.6 1.3 0.4 0.1 123N123S 0.9 1.3 1.2 1.1 0.2 0.1 124 M124A 0.6 1.4 1.0 1.0 1.0 0.7 124M124H 0.2 1.4 0.3 0.3

124 M124N 0.2 3.0 0.8 0.8

124 M124Q 0.2 5.2 2.6 1.4

124 M124S 0.3 2.8 1.4 1.5 0.9 0.1 124 M124T 0.5 1.8 1.3 1.4 0.9 0.2 124M124V 1.1 1.4 1.2 1.1 0.9 0.3 125 S125A 0.9 1.9 1.4 1.2 1.1 0.1 126L126A 0.9 1.6 1.1 1.2

126 L126Q 0.3 3.1 0.9 1.1

126 L126S 0.7 1.6 0.9 0.9

126 L126T 0.5 2.4 1.5 1.4

126 L126Y 0.6 1.5 1.1 1.3

128 G128E 0.3 1.6 1.2 1.5

128 G128N 0.6 1.6 1.6 1.3

128 G128T 0.3 2.2 2.4 1.6

129 P129V 0.7 1.4 1.3 1.1 0.8 0.8 130 S130P 0.6 1.3 1.3 1.0 0.7 0.1 132S132I 0.3 1.6 1.8 1.3 0.8 0.6 132 S132N 0.9 1.4 1.4 1.2 1.0 0.9 132S132P 0.2 9.4 11.7  2.5 1.0 0.2 132 S132Q 0.3 1.4 1.3 1.2 0.7 0.9 132S132V 0.2 3.3 2.4 2.1 0.8 0.6 134 A134I 0.3 1.6 1.2 1.3 0.9 0.9 134A134L 0.2 1.9 2.4 1.8 1.1 0.6 134 A134M 0.2 1.4 1.2 1.3 1.1 0.5 135L135I 0.3 1.4 1.0 1.1 0.7 0.2 135 L135T 0.2 3.4 2.3 2.1 0.6 0.1 135L135V 0.3 2.1 1.1 1.3 0.6 0.2 135 L135W 0.3 2.1 2.0 1.5 0.8 0.2 135L135Y 0.3 1.9 2.0 1.7

136 K136D 0.2 1.6 1.5 0.9 0.4 0.5 136 K136F 0.3 1.4 1.6 1.2 0.6 0.8 136K136I 0.2 2.2 2.5 1.4 0.4 0.5 136 K136V 0.2 2.2 2.4 2.0 0.5 0.8 136K136Y 0.3 1.8 1.4 1.2 0.4 0.9 137 A137P 0.2 3.9 3.4 1.8 0.2 0.1 138A138D 0.2 1.5 4.6 2.3 0.3 0.2 138 A138E 0.4 1.4 1.0 1.1 0.9 0.8 138A138F 0.2 2.6 2.7 2.0 0.8 0.3 138 A138H 0.2 2.0 2.2 1.8 0.7 0.5 138A138Q 0.2 11.9 16.2  5.0 0.9 0.8 138 A138Y 0.2 6.0 9.1 3.3 0.6 0.5 142A142G 0.5 1.3 1.2 1.0 1.0 1.2 142 A142I 0.3 1.6 1.4 1.5 0.6 0.8 142A142T 0.3 1.3 1.6 1.5 0.8 1.1 142 A142V 0.4 1.5 1.2 1.2 0.8 1.1 143V143W 0.7 1.4 0.8 1.0 0.9 0.9 144 A144P 0.2 1.8 2.6 1.2 0.4 0.3 146G146L 0.2 2.2 3.8 1.9 1.1 0.6 146 G146T 0.3 1.5 1.8 1.6 0.7 1.1 146G146Y 0.2 1.7 2.2 1.6 0.9 0.9 147 V147D 0.2 2.3 3.1 2.1 1.2 0.5 147V147P 0.3 1.7 2.1 1.5 0.8 1.1 147 V147W 0.3 2.0 2.2 1.3 0.9 0.9 147V147Y 0.3 2.3 2.3 1.5 0.9 0.8 148 V148N 0.4 1.4 1.4 1.1 0.8 1.1 148V148Y 0.2 1.6 1.5 1.6 0.6 0.4 149 V149E 0.2 2.7 3.7 2.5 1.0 0.4 151A151T 0.5 1.4 1.2 1.2 0.6 0.6 153 A153T 0.2 4.4 4.3 3.0

0.3 153 A153V 0.3 1.3 1.2 1.4 0.3 0.9 159 S159K 1.2 1.4 0.9 1.0 1.0 1.1164 T164W 0.2 1.6 2.3 1.7 0.1 0.2 165 V165T 0.2 1.5 2.0 1.9 0.1 0.2 167Y167A 0.7 1.4 1.1 1.3 0.7 0.6 167 Y167D 0.3 1.4 1.6 1.3 0.1 0.1 167Y167E 0.4 1.5 1.7 1.3 0.5 0.1 167 Y167M 0.2 1.7 1.3 1.4 0.4 0.2 167Y167P 0.3 1.8 1.9 1.6

167 Y167S 0.3 1.4 1.3 1.3 0.2 0.3 167 Y167T 0.3 1.5 1.3 1.4 0.3 0.4 168P168L 0.2 2.0 0.9 0.3

168 P168T 0.2 3.0 0.3

169 G169C 0.2 3.0 2.2 2.4 1.0 0.5 170 K170E 0.2 2.9 0.1 1.2

170 K170N 0.3 1.6 1.4 1.2 0.5 0.6 170 K170P 0.2 2.5 2.6 1.6 0.9 0.7 170K170Q 0.5 1.3 1.2 1.1 0.8 0.6 170 K170S 0.5 1.3 1.1 0.9 0.9 0.8 170K170T 0.3 1.5 1.5 1.1 0.5 0.5 170 K170Y 0.4 1.4 1.2 1.0 0.9 0.7 171Y171C 0.2 12.3 8.6 1.8 0.8 0.1 171 Y171D 0.2 4.6 3.5 1.1

171 Y171L 0.2 4.1 3.4 2.4 0.3 0.3 171 Y171N 0.2 2.2

0.5

171 Y171W 0.5 1.4 1.0 1.0 0.6 0.4 172 P172E 0.8 1.3 1.2 1.0 1.2 1.0 172P172G 0.3 1.4 1.2 1.2 1.0 0.9 172 P172I 0.5 1.4 1.1 1.1 0.9 1.1 172P172L 0.5 1.5 1.1 1.1 1.0 1.0 172 P172V 0.5 1.4 1.2 1.2 0.8 1.2 172P172Y 0.3 1.7 1.3 1.4 0.8 0.9 173 S173H 0.5 1.4 0.9 1.2 0.8 0.9 173S173W 0.3 1.9 1.3 1.5 0.6 0.9 173 S173Y 0.4 1.7 1.0 1.4 0.7 0.9 174V174A 0.5 1.4 1.2 1.1 0.8 1.0 174 V174I 0.4 1.5 0.9 1.1 0.6 0.6 174V174L 0.2 93.0 66.8  4.2 0.5 0.3 174 V174S 0.4 1.4 1.0 1.2 0.9 0.8 175I175A 0.2 6.1 9.3 1.8 0.4 0.3 175 I175F 0.3 1.7 1.2 1.3 0.6 0.8 175I175R 0.2 1.5

0.1

175 I175T 0.3 2.0 1.6 1.4 0.5 0.9 175 I175V 0.3 2.0 1.3 1.4 0.9 1.1 176A176V 0.2 1.5

0.6

177 V177W 0.2 3.8 5.4 4.5 0.2 0.5 180 V180F 0.2 1.7

1.5

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0.3 182 S182W 0.3 1.5 1.4 1.4 0.3 1.0 184 N184A 0.3 1.4 1.9 1.4 0.7 0.7184 N184L 1.0 1.4 0.9 1.1 0.7 0.9 184 N184V 0.7 1.3 1.1 1.0 0.3 1.0 185Q185D 0.3 2.0 1.7 1.4 0.6 0.6 185 Q185E 0.8 1.4 1.1 0.9 1.2 1.0 185Q185I 0.9 1.5 1.0 1.0 0.9 1.2 185 Q185S 0.8 1.4 1.0 1.1 0.8 1.1 185Q185T 0.8 1.4 1.3 1.1 1.0 1.2 186 R186C 0.4 1.9 1.6 1.1 0.6 0.5 186R186D 0.2 11.1 16.4  0.2

186 R186E 0.2 23.1 23.9  1.8 0.1 0.1 186 R186F 0.3 1.3 1.4 0.9 0.2 0.3186 R186G 0.3 2.2 1.9 1.2 0.2 0.6 186 R186I 0.4 1.3 2.1 1.3 0.9 0.8 186R186L 0.5 1.6 1.4 1.2 0.9 0.9 186 R186N 0.3 2.5 3.7 1.5 0.3 0.4 186R186P 0.2 3.0 2.3 1.0 0.1 0.2 186 R186Q 0.4 1.5 2.1 1.3 0.8 0.9 186R186S 0.3 2.4 1.9 1.4 0.3 0.4 186 R186T 0.2 8.6 7.0 2.7 0.4 0.4 186R186V 0.4 2.0 2.0 1.4 0.9 0.8 186 R186W 0.6 1.5 1.7 1.1 1.1 1.0 186R186Y 0.4 1.5 2.0 1.4 0.8 0.7 187 A187D 0.9 1.5 1.0 1.0 0.4 0.2 187A187E 0.7 1.4 1.5 1.0 0.2 0.3 187 A187Q 0.5 1.3 1.2 0.9

0.4 190 S190G 0.3 1.5 2.2 1.3 0.1 0.6 190 S190N 0.2 1.6 2.0 1.1 0.1 0.1192 Y192D 0.2 1.9 2.2 1.2 0.1 0.1 192 Y192E 0.4 1.4 1.6 1.1

0.2 192 Y192L 0.2 1.9 1.9 1.5

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193 G193Q 0.2 2.4 0.1 0.1

193 G193T 0.2 4.8 0.5 0.1

193 G193V 0.2 2.3 1.2

194 P194E 0.7 1.5 1.1 1.0 1.0 1.3 194 P194I 0.5 1.6 1.1 1.2 0.9 1.1 194P194L 0.6 1.6 0.9 1.3 0.9 1.1 194 P194M 0.6 1.4 1.1 1.1 0.8 1.5 194P194N 0.5 1.8 1.0 1.2 0.1 1.7 194 P194T 0.6 1.3 1.4 1.1 0.9 1.2 194P194V 0.5 1.7 1.1 1.2 0.8 1.4 195 E195C 0.3 1.4 0.9 0.6 0.4 0.2 195E195K 0.2 3.7 2.6 2.2 0.3 0.5 195 E195W 0.2 4.3 1.3 1.3 0.1 0.1 196L196I 0.4 1.7 1.2 1.2 0.8 1.0 196 L196M 0.4 1.5 1.0 1.1 0.2 0.7 196L196T 0.2 3.9 2.9 2.7 1.0 0.7 196 L196V 0.3 2.0 1.3 1.5 0.7 0.8 197D197I 0.2 3.1 4.1 2.6

0.3 197 D197M 0.2 1.5 0.7 1.1 0.1 0.1 199 M199F 0.2 28.7 28.8  2.6 0.10.2 199 M199Q 0.2 2.1 1.7 1.7 0.8 0.6 200 A200C 0.7 1.3 0.9 1.2 0.6 1.2200 A200H 0.2 5.0

0.2

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0.4 200 A200Y 0.2 2.6

0.1

201 P201L 0.3 1.7 1.7 2.1

0.6 201 P201T 0.2 4.0 4.1 3.5

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0.5 205 I205T 0.8 1.6 1.2 1.1 0.8 1.3 208 T208C 0.8 1.5 1.1 1.2 0.8 1.1208 T208L 0.9 1.4 1.1 1.1

1.2 208 T208M 0.2 5.7 6.0 1.8 0.4 0.4 208 T208P 0.4 1.7 1.1 1.2

0.7 208 T208V 0.6 1.3 1.4 1.2

0.9 209 L209C 0.8 1.4 1.0 1.1 0.9 1.5 209 L209W 0.8 1.4 1.0 1.1 0.3 1.3210 P210C 0.4 1.5 0.9 1.0 0.2 0.8 210 P210D 0.3 1.4 0.8 1.2 0.1 0.3 210P210E 0.4 1.7 1.1 1.3 0.4 0.7 210 P210F 0.2 10.3 3.5 2.0 0.1 0.1 210P210G 0.3 1.8 1.0 1.3

0.6 210 P210Q 0.3 3.0 2.3 2.0

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0.7 214 Y214S 0.2 2.2 1.1 1.5 0.2 0.1 215 G215D 0.9 1.3 1.1 1.1 1.0 0.7215 G215Q 0.8 1.4 1.0 1.0 0.1 0.7 215 G215V 0.4 1.5 0.9 1.2 0.1 0.5 216A216E 1.2 1.4 0.9 1.0 1.4 0.8 217 Y217E 1.0 1.4 1.0 1.2 1.3 0.5 217Y217L 1.0 1.3 1.0 1.1 1.4 3.5 217 Y217M 0.9 1.3 1.2 1.1 1.5 1.8 218N218P 0.3 1.6 0.8 1.2 0.2 0.6 223 A223W 0.2 1.5 0.8 0.2

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0.1

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0.5 254 T254Q 0.2 32.6 41.1  3.7

0.5 254 T254R 0.2 7.7 3.7 1.6

0.6 255 T255L 1.0 1.5 1.0 1.0

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0.4 256 K256G 0.7 1.3 0.9 1.0

1.0 256 K256R 1.1 1.4 1.0 1.1

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0.7 263 Y263K 0.2 2.4 2.8 1.9 0.1 0.3 263 Y263R 0.2 8.0 4.1 1.7 0.1 0.3265 K265P 0.3 1.6 1.9 1.6 1.0 0.8 268 I268S 0.3 1.4 1.3 1.6

0.8 268 I268T 0.3 1.9 2.0 1.8 0.8 0.4 268 I268W 0.2 2.8 2.4 0.2

270 V270F 0.2 2.4 2.9 1.1 0.2 0.1 273 A273K 0.2 1.6 1.6 1.9

0.6 273 A273P 0.3 1.5 1.7 1.8

0.7 273 A273R 0.2 1.4 1.2 2.0

0.6 273 A273V 0.5 1.4 1.2 1.2 0.3 0.8 273 A273W 0.3 2.1 2.0 1.5

0.8 274 A274W 0.4 1.3 0.7 1.1 0.5 0.7Table 31-4

-   Parent: FNA (SEQ ID NO:2 with Y217L mutation)-   Measure: PI versus FNA for BMI pH8 16C assay-   Detergent: As indicated in table 31-1 (NA Detergent was Commercial    heat deactivated Tide 2×; WE detergent was commercial heat    deactivated Western European Henkel Persil liquid laundry and Japan    laundry was heat deactivated Japanese Tide laundry powder    detergent).    Generation of Combinatorial Variants of BPN′-Y217L Involving Charged    Residue Substitutions

The pAC-FNAre plasmid in FIG. 9 containing the BPN′-Y217L gene was sentto DNA 2.0 Inc. (Menlo Park, Calif.) for the generation of combinatorialvariants containing substitutions involving charge changes. They werealso provided with the Bacillus subtilis strain (genotype: ΔaprE, ΔnprE,ΔspoIIE, amyE::xylRPxylAcomK-phleo) for transformations. The subtilisinBPN′-Y217L combinatorial charge variants were designed by identifyingfour well-distributed, surface-exposed, uncharged polar amino-acidresidues outside the active site. These residues are Ser-87, Asn-109,Ser-188, and Ser-248. The variants were supplied as glycerol stocks fromDNA 2.0. B. subtilis transformants containing BPN′ variant proteins werereplicated with a steel 96-well replicator from glycerol stocks into96-well culture plates (BD, 353075) containing 200 μl of LB media+25μg/ml chloramphenicol, grown overnight at 37° C., 220 rpm in ahumidified enclosure. 200 μl from the overnight culture was used toinoculate 2000 μl MBD defined media+25 μg/ml chloramphenicol in 5 mlplastic shake tubes. MBD (a MOPS based defined medium) was madeessentially as known in the art (See, Neidhardt et al., J. Bacteriol.,119: 736-747 [1974]), except that NH₄Cl, FeSO₄, and CaCl₂ were omittedfrom the base medium, 3 mM K₂HPO₄ was used, and the base medium wassupplemented with 60 mM urea, 75 g/L glucose, and 1 soytone. Themicronutrients were made up as a 100× stock solution containing in oneliter, 400 mg FeSO₄ 7H₂O, 100 mg MnSO₄.H₂O, 100 mg ZnSO₄ 7H₂O, 50 mgCuCl₂ 2H₂O, 100 mg CoCl₂ 6H₂O, 100 mg NaMoO₄ 2H₂O, 100 mg Na₂B₄O₇ 10H₂O,10 ml of 1M CaCl₂, and 10 ml of 0.5 M sodium citrate. Shake tubes wereincubated at 37° C., 220 rpm, for 60 hours. Following 60 hours, culturebroth was spun down in a centrifuge at greater than 8000×RCF. Thesupernatant solution was decanted into 15 ml polypropylene conical tubesfor storage. No further purification or concentration was performed.Supernatant stocks were formulated to 40% propylene glycol for long-termstability.

Description

This example describes the testing of BPN′-Y217L variants in BMImicroswatch assays in detergents representing various market geographies(e.g., differing pH, T, and/or water hardness), in laundry applications,as described in Test Method 2.

TABLE 31-4 Performance of combinatorial variants of BPN′-Y217L involvingcharged residue substitutions Net Laundry PI WE Laundry Variants ChargePI NA Laundry PI JPN N109D-Y217L-S248R 0 1.2 1.9 1.6 N109D-S188R-Y217L 01.3 1.5 1.3 S87D-Y217L-S248R 0 1.2 1.5 1.5 S87R-N109D-Y217L-S248R 1 1.21.5 2.3 S87R-N109D-S188D-Y217L- 0 1.2 1.5 1.0 S248RTable 31-5: Wash Performance of Selected Double Mutant Proteases

-   Parent: BPN′ wild type (SEQ ID NO: 2)-   Measure: PI at least 1.3 for BMI assay pH8 16C versus wild type (SEQ    ID NO:2)-   Detergent: Commercial heat deactivated Tide 2× Cold    Description

The BPN′ double mutants shown in Table 31-5 below were produced by DNA2.0 (Menlo Park, Calif.), using BPN′ as the parent gene contained in theexpression plasmid pHPLT-BPN′ partial opt. Plasmid pHPLT-BPN′ partialopt was also created by DNA 2.0 (Menlo Park, Calif.) using a codonoptimized gene encoding the BPN′ protease and the pHPLT-BPN′ plasmiddescribed above (also see FIG. 3).

The DNA sequence of the pHPLT-BPN′ expression cassette (aprE-BPN′ hybridleader, BPN′ pro and BPN′ mature DNA sequence from B. amyloliquefaciens)provided below, encodes the BPN′ precursor protein:

GTGAGAAGCAAAAAATTGTGGATCAGTTTGCTGTTTGCTTTAGCGTTAATCTTTACGATGGCGTTCGGCAGCACATCCTCTGCCCAGGCGGCAGGGAAATCAAACGGGGAAAAGAAATATATTGTCGGGTTTAAACAGACAATGAGCACGATGAGCGCCGCTAAGAAGAAAGATGTCATTTCTGAAAAAGGCGGGAAAGTGCAAAAGCAATTCAAATATGTAGACGCAGCTTCAGCTACATTAAACGAAAAAGCTGTAAAAGAATTGAAAAAAGACCCGAGCGTCGCTTACGTTGAAGAA GATCACGTAGCACATGCGTACGCGCAGTCCGTGCCTTACGGCGTATCACAAATTAAAGCCCCTGCTCTGCACTCTCAAGGCTACACTGGATCAAATGTTAAAGTAGCGGTTATCGACAGCGGTATCGACTCGAGCCATCCAGATCTTAAAGTCGCTGGAGGGGCTTCTATGGTGCCGTCCGAAACAAACCCGTTTCAAGATAACAATTCTCATGGCACACACGTCGCAGGAACGGTTGCGGCGTTAAACAATTCTATTGGCGTGCTTGGTGTAGCCCCGTCTGCTTCGCTCTACGCCGTTAAAGTTCTTGGCGCAGACGGATCAGGCCAATACTCATGGATTATCAACGGCATCGAATGGGCCATCGCGAATAACATGGATGTAATCAACATGAGCCTGGGAGGACCAAGCGGCAGTGCGGCACTTAAAGCAGCAGTTGATAAAGCTGTTGCATCTGGTGTCGTCGTAGTAGCGGCAGCTGGGAATGAGGGAACATCCGGATCATCGAGTACCGTCGGTTATCCAGGCAAGTACCCTTCAGTGATTGCAGTGGGCGCTGTAGACTCTTCAAATCAACGTGCCTCTTTTTCCTCCGTGGGACCGGAGCTGGATGTCATGGCCCCTGGCGTTTCTATTCAATCGACGCTTCCAGGGAACAAGTATGGTGCGTATAACGGGACTTCCATGGCCTCGCCGCATGTAGCTGGGGCGGCCGCATTGATTCTTTCTAAGCACCCGAACTGGACAAACACTCAAGTCCGCAGCAGTTTAGAAAACACCACTACAAAACTTGGTGATTCTTTCTACTATGGAAAAGGGCTGATCAACGTACAGGCGGCAGCTCAG

In the above sequence, bold indicates the DNA that encodes the matureprotease, standard font indicates the leader sequence (aprE-BPN′ hybridleader), and the underlined indicates the pro sequences (BPN′). Theamino acid sequence of the mature BPN′ protease is SEQ ID NO:2. Theplasmid map

The BPN′ variant proteins were also produced as described earlier.Protein concentration of culture supernatants was determined by TCAprecipitation as described in TCA assay of Test Method 2. The stainremoval performance of the variants was tested in laundry applicationson EMPA 116 swatches (BMI stain, CFT) at pH 8/16° C., pH 7/16° C. and pH8/32° C. using methods described in Test Method 2 assay methods, withthe following modifications. The test detergent used was heatinactivated TIDE® 2× Cold detergent (Proctor & Gamble). Heatinactivation of commercial detergent formulas serves to destroy theendogenous enzymatic activity of any protein components while retainingthe properties of nonenzymatic components. Heat inactivation of thedetergents was performed by placing pre-weighed amounts of liquiddetergent (in a glass bottle) in a water bath at 95° C. for 2 hours. Thedetergent was purchased from local supermarket stores. Both unheated andheated detergents were assayed within 5 minutes of dissolving thedetergent, in order to accurately determine percentage deactivated.Functionality of BPN′ variants was quantified as a performance index(PI) (i.e., the ratio of performance of a variant relative to parentBPN′). Results are shown in Table 31-5—in this Table, “ND” indicates“not determined.” Data are shown for variants having a PI of at least1.3 for BMI pH8 16C cleaning assay.

TABLE 31-5 PI Values of BPN′ Variants Tested for Expression (TCA) andStain Removal Performance (BMI pH 8/16° C., BMI pH 7/16° C., and BMI pH8/32° C.) BMI BMI BMI pH 8/ pH pH Variant TCA 16° C. 7/16° C. 8/32° C.G128A-Y217Q 1.2 1.4 1.1 1.2 I111V-M124V 0.9 1.3 ND ND M124V-Y217Q 1.51.4 1.1 1.2 N62Q-G97A 1.5 1.3 1.2 1.2 S89Y-M124V 1.1 1.3 ND ND V68A-A92G0.8 1.4 ND ND V68A-G97A 2.1 1.3 ND ND V68A-I111V 2.1 1.3 ND ND V68A-S89Y1.7 1.4 ND ND V68A-V227T 1.0 1.4 ND ND V68A-Y217Q 2.3 1.3 ND NDW106F-Y217Q 0.9 1.4 ND NDTable 31-6: Wash Performance of Selected Series 1 BPN′ Cold WaterProteases

-   Parent: BPN′ wild type (SEQ ID NO: 2)-   Measure: PI at least 1.3 for BMI assay pH8 16C versus wild type (SEQ    ID NO:2)-   Detergent: Commercial heat deactivated Tide 2× Cold    Description

BPN′ multiple mutation libraries (or combinatorial libraries) wereproduced by Geneart or DNA 2.0, using BPN′ as the parent protein.Protein concentration of culture supernatants was determined by TCAprecipitation as described in Test Method 2. The stain removalperformance of the variants was tested in laundry applications on EMPA116 swatches (BMI stain, CFT) at pH 8/16° C., pH 7/16° C. and pH 8/32°C. using methods described in Test Method 2, with the followingmodifications. The test detergent used was heat inactivated TIDE® 2×Cold detergent (Proctor & Gamble), prepared as described in Test Method2. Heat inactivation of commercial detergent formulas serves to destroythe endogenous enzymatic activity of any protein components whileretaining the properties of non-enzymatic components. Heat inactivationof the detergents was performed by placing pre-weighed amounts of liquiddetergent (in a glass bottle) in a water bath at 95° C. for 2 hours. Thedetergent was purchased from local supermarket stores. Both unheated andheated detergents were assayed within 5 minutes of dissolving thedetergent, in order to accurately determine percentage deactivated.Enzyme activity was tested by AAPF assay. Functionality of BPN′ variantswas quantified as a performance index (PI) (i.e., the ratio ofperformance of a variant relative to parent BPN′). Results are shown inTable 31-6. Performance indices less than or equal to 0.1 were fixed to0.1 and indicated in bold italics in the table. For every variant with aTCA protein performance index less than or equal to 0.1, all values werefixed at 0.1.

TABLE 31-6 PI Values of BPN′ Variants Tested for Expression (TCA) andStain Removal Performance (BMI pH 8/16° C., BMI pH 7/16° C., and BMI pH8/32° C.) BMI BMI BMI pH pH pH Variant TCA 8/16° C. 7/16° C. 8/32° C.G97A-G128A-Y217Q 1.3 1.4 1.2 1.2 G97A-L126A-Y217Q 1.1 1.3 1.2 1.2G97A-M124V-L126A-Y217Q 1.8 1.4 1.2 1.2 G97A-N123G-Y217Q 0.6 1.4 1.2 1.1L96T-G97A-Y217Q 0.5 1.3 1.1 1.1 M124V-L126A-Y217Q 1.7 1.4 1.2 1.2N62Q-G128A-Y217Q 0.7 1.3 1.2 1.1 N62Q-G97A-Y217Q 1.7 1.4 1.2 1.3Table 31-7: Wash Performance of Selected Series 1 BPN′ Cold WaterProtease Variants

-   Parent: FNA (SEQ ID NO:2 with Y217L mutation)-   Measure: PI versus FNA for BMI pH8 16C assay-   Detergent: Commercial heat deactivated Tide 2× Cold    Description

Saturation libraries at positions 97-128-217 in BPN′ (parent) wereproduced by DNA 2.0. Protein concentration of culture supernatants wasdetermined by TCA precipitation as described in Test Method 2. The stainremoval performance of the variants was tested in laundry applicationson EMPA 116 swatches (BMI stain, CFT) at pH8/16° C. using methodsdescribed in Test Method 2. Functionality of BPN′ variants wasquantified as a performance index (PI) (i.e., the ratio of performanceof a variant relative to FNA). Results are shown in Table 31-7.

TABLE 31-7 PI Values of BPN′ Variants Tested for Protein Determination(TCA) and Stain Removal Performance (BMI pH 8/16° C.) Variants TCA BMIpH 8 16° C. G97N-G128A-Y217M 1.1 1.4 G97G-G128S-Y217E 1.5 1.4G97A-G128A-Y217Q 1.3 1.4 G97M-G128S-Y217E 1.2 1.4 G97A-G128S-Y217Q 1.91.3 G97D-G128S-Y217Q 1.6 1.3 G97M-G128G-Y217M 1.6 1.3 G97G-G128S-Y217Q1.6 1.3 G97S-G128S-Y217Q 1.5 1.3 G97G-G128A-Y217Q 1.3 1.3G97S-G128A-Y217E 1.0 1.3 G97A-G128S-Y217L 2.2 1.3 G97A-G128A-Y217N 1.21.3 G97Q-G128S-Y217L 1.9 1.3 G97A-G128A-Y217M 1.5 1.3 G97A-G128A-Y217S1.4 1.3 G97D-G128A-Y217Q 1.1 1.3 G97M-G128S-Y217Q 1.0 1.3G97Q-G128G-Y217D-S87Y 1.5 1.3 G97S-G128A-Y217N 1.1 1.3 G97A-G128S-Y217T1.6 1.3 G97D-G128S-Y217E 1.0 1.3 G97D-G128A-Y217L 1.4 1.3G97G-G128S-Y217E-S78P-A272T 1.0 1.3 G97T-G128S-Y217D 1.1 1.3G97D-G128A-Y217I 1.0 1.3 G97Q-G128S-Y217Q 1.6 1.3 G97G-G128A-Y217D 1.11.3 G97Q-G128A-Y217N 1.1 1.3 G97S-G128A-Y217M 1.4 1.3 G97S-G128S-Y217N1.6 1.3 G97S-G128S-Y217M 1.5 1.3 G97E-G128S-Y217M 1.6 1.2G97S-G128P-Y217Q 1.0 1.2 G97T-G128S-Y217Q 1.1 1.2 G97D-G128S-Y217Q-A73T1.2 1.2 G97E-G128S-Y217N 1.2 1.2 G97G-G128A-Y217I 1.5 1.2G97Q-G128A-Y217D 1.1 1.2 G97Q-G128S-Y217M 2.0 1.2 G97R-G128T-Y217Q-S162P0.7 1.2 G97S-G128S-Y217D 1.5 1.2 G97T-G128P-Y217I 1.3 1.2G97Q-G128G-Y217E 1.6 1.2 G97C-G128G-Y217N 1.3 1.2 G97D-G128S-Y217H 1.51.2 G97M-G128S-Y217L 1.0 1.2 G97M-G128S-Y217N 1.0 1.2 G97S-G128S-Y217E0.6 1.2 G97M-G128S-Y217I 1.1 1.2 G97A-G128P-Y217A 0.8 1.2G97R-G128S-Y217D 1.0 1.2Table 31-8: Wash Performance of Selected Series 1 BPN′ Cold WaterProtease Variants

-   Parent: BPN′ v3 (SEQ ID NO:4)-   Measure: PI versus above parent using BMI pH8 16° C. assay-   Detergent: Commercial heat deactivated Tide 2× Cold    Description

Additional BPN′ multiple mutation libraries were produced by Geneart orGene Oracle, using BPN′: G97A-G128A-Y217Q protein as the parentmolecule. Results of experiments conducted to determine stain removalactivity (microswatch assay to determine stain removal performance inlaundry applications using EMPA 116 swatches (BMI stain, CFTVlaardingen) (BMI pH8, BMI pH7, BMI 32° C.), protein determination byTCA precipitation, and LAS/EDTA stability (tests of properties ofinterest) of BPN′ variants are shown in table 31-8. The results wereobtained using the methods described in Test Method 2, with thefollowing modifications for the stain removal performance assay. Thetest detergent used was heat inactivated TIDE® 2× Cold detergent(Proctor & Gamble, Cincinnati, Ohio, USA). Heat inactivation ofcommercial detergent formulas serves to destroy the endogenous enzymaticactivity of any protein components while retaining the properties ofnon-enzymatic components. Heat inactivation of the detergents wasperformed by placing pre-weighed amounts of liquid detergent (in a glassbottle) in a water bath at 95° C. for 2 hours. The detergent waspurchased from local supermarket stores. Both unheated and heateddetergents were assayed within 5 minutes of dissolving the detergent toaccurately determine percentage deactivated. Enzyme activity was testedby AAPF assay. As described throughout herein, functionality of BPN′variants was quantified as a performance index (PI), which is the ratioof performance of a variant to parent protein BPN′: G97A-G128A-Y217Q(i.e. enzyme of SEQ ID NO:4).

TABLE 31-8 Stain removal performance of multiple mutation variants ofBPN′ BMI BMI BMI pH 8 pH 7 pH 8 Variant 16 C. 16 C. 32° C. TCAG97A-G128A-Y217Q-S145D 1.1 1.0 1.2 1.0 G97A-G128A-Y217Q-P239R 1.0 1.01.1 0.7 G97A-G128A-Y217Q-N61E-P129E- 1.0 0.9 1.1 1.0 S162K-K213L-N240KG97A-G128A-Y217Q-N61E 1.0 1.0 1.1 1.3 G97A-G128A-Y217Q-P40E-A144K- 1.00.9 0.9 0.8 K213L G97A-G128A-Y217Q-P129E 1.0 1.0 1.0 1.0G97A-G128A-Y217Q-N61E-P129E- 1.0 1.0 1.1 1.2 S159KG97A-G128A-Y217Q-K213L 1.0 1.0 1.0 1.1 G97A-G128A-Y217Q-S87D 1.0 1.0 1.00.8 G97A-G128A-Y217Q-Q206E 1.0 1.0 1.0 1.0 G97A-G128A-Y217Q-S24R-P40E-1.0 1.0 1.0 0.9 S145D-S159K-K213L G97A-G128A-Y217Q-K265N 1.0 1.0 1.0 0.9G97A-G128A-Y217Q-S24R 1.0 0.9 1.0 1.0 G97A-G128A-Y217Q-P40E 1.0 1.0 1.00.8 G97A-G128A-Y217Q-Q275E 1.0 1.0 1.0 1.0 G97A-G128A-Y217Q-P129E-S145D-1.0 1.0 1.0 0.8 N240K G97A-G128A-Y217Q-A144K 1.0 0.9 1.0 0.9G97A-G128A-Y217Q-S159K 0.9 0.9 1.0 1.0 G97A-G128A-Y217Q-S162K 0.9 0.91.0 1.0 G97A-G128A-Y217Q-N240K 0.9 0.9 1.0 0.7Table 31-9: Wash Performance of Selected Series 1 BPN′ Cold WaterProtease Variants

-   Parent: BPN′ v3 (G97A-G128A-Y217Q)-   Measure: PI relative to BPN′ v3 (G97A-G128A-Y217Q) in BMI pH8 16C    assay-   Detergent: Commercial heat deactivated Tide 2× Cold    Description

Additional BPN′ multiple mutation libraries were produced by Geneart orGene Oracle, using BPN′: G97A-G128A-Y217Q protein as the parentmolecule. Results of experiments conducted to determine stain removalactivity (microswatch assay to determine stain removal performance inlaundry applications using EMPA 116 swatches (BMI stain, CFTVlaardingen) (BMI pH8, BMI pH7, BMI 32° C.), protein determination byTCA precipitation, and LAS/EDTA stability (tests of properties ofinterest) of BPN′ variants are shown in table 31-9. The results wereobtained using the methods described in Test method 2, with thefollowing modifications for the stain removal performance assay. Thetest detergent used was heat inactivated TIDE® 2× Cold detergent(Proctor & Gamble, Cincinnati, Ohio, USA). Heat inactivation ofcommercial detergent formulas serves to destroy the endogenous enzymaticactivity of any protein components while retaining the properties ofnon-enzymatic components. Heat inactivation of the detergents wasperformed by placing pre-weighed amounts of liquid detergent (in a glassbottle) in a water bath at 95° C. for 2 hours. The detergent waspurchased from local supermarket stores. Both unheated and heateddetergents were assayed within 5 minutes of dissolving the detergent toaccurately determine percentage deactivated. Enzyme activity was testedby AAPF assay. As described throughout herein, functionality of BPN′variants was quantified as a performance index (PI), which is the ratioof performance of a variant to parent protein BPN′: G97A-G128A-Y217Q(i.e. enzyme of SEQ ID NO:4).

TABLE 31-9 Stain removal performance of multiple mutation variants ofBPN′ BMI BMI LAS/ Mutations pH8 pH7 TCA EDTA G97A-G128A-Y217Q-S53G 1.11.0 1.2 1.0 G97A-G128A-Y217Q-S78N 1.0 1.0 1.0 1.5G97A-G128A-Y217Q-S53G-S78N 1.0 1.0 1.1 1.4 G97A-G128A-Y217Q-S53G-I111V1.0 1.0 0.8 1.2 G97A-G128A-Y217Q-S53G-N117S 0.9 1.0 0.8 1.3G97A-G128A-Y217Q-S53G-S132N 0.9 0.9 0.7 1.0 G97A-G128A-Y217Q-Y104N-S132N1.0 1.1 0.7 1.3 G97A-G128A-Y217Q-S53G-S78N-I111V 1.0 1.0 0.9 1.7G97A-G128A-Y217Q-S53G-S78N-N117S 0.9 0.9 0.7 1.6G97A-G128A-Y217Q-S53G-S78N-S132N 1.0 0.9 0.8 1.5G97A-G128A-Y217Q-S53G-Y104N- 0.9 1.0 1.2 1.1 S132NG97A-G128A-Y217Q-S78N-Y104N- 1.0 1.0 0.8 N/D S132NTable 31-10

-   Parent FNA (BPN′ Y217L)-   Measure PI relative to FNA (BPN′ Y217L) for BMI pH8 16° C. assay    Detergent: Commercial heat-deactivated Tide 2×    Targeted ISD (Insertion Substitution Deletion) Library Construction

A PCR-based method was used to create a library of modified B.amyloliquefaciens subtilisin BPN′-Y217L (commercially available asPURAFECT® PRIME subtilisin) polynucleotides containing in-frameinsertions, deletions and/or substitutions in two regions of BPN′-Y217L(positions 63-77 and 92-132 of the mature region) as shown in FIG. 10.This method is basically identical to the described in Pisarchik et al.,Prot. Eng. Des. Select., 20:257-265 [2007] with several minorvariations. First, we used EamI restriction enzyme to generate stickyends in our PCR fragments instead of KasI. We also introduced degeneratecodon (NDT) right next to the sticky end. Finally, we placed primersmuch more densely with every following primer being moved only 3 bpdownstream until the end of the targeted region was reached. Two sets ofoligonucleotides that evenly cover the targeted regions of theBPN′-Y217L gene of a full-length protein of 392 amino acids, in bothforward and reverse direction were used to amplify the 5′ and 3′segments of the portion of the BPN′-Y217L gene. The coding region of theBPN′-Y217L mature protease contains the KpnI and XhoI restriction sitesfor cloning purposes:

gtgagaagcaaaaaattgtggatca gtttgctgtttgctttagcgttaatctttacgatggcgttcggcagcacatccagcgcgcaggctgcagggaaatcaaacggggaaaagaaatatattgtcgggtttaaacagacaatgagcacgatgagcgccgctaagaagaaagacgtcatttctgaaaaaggcgggaaagtgcaaaagcaattcaaatatgtagacgcagctagcgctacattaaacgaaaaagctgtaaaagaattgaaaaaagacccgagcgtcgcttacgttgaagaagatcacgtagcacacgcgtac gcgcagtccgtgccatatggcgtatcacaaattaaagcccctgctctgcactctcaaggctacaccggttcaaatgttaaagtagcggttatcgacagcggtatcgattcttctcatccagatcttaaagtagcaggcggagccagcatggttccttctgaaacaaatcctttccaagacaacaactctcacggaacacacgttgctggtaccgttgcggctcttaataactcaatcggtgtattaggcgttgcgccaagcgcatcactttacgctgtaaaagttctcggcgccgacggttccggccaatacagctggatcattaacggaatcgagtgggcgatcgcaaacaatatggacgttattaacatgagcctcggcggaccgtccggttctgctgctttaaaagcggcagttgataaagccgttgcatccggcgtcgtagtcgttgcggcagccggcaacgaaggcacttccggcagctcaagcacagtgggctaccctggtaaatacccttctgtcattgcagtaggcgctgtcgacagcagcaaccaaagagcatctttctcaagcgtaggacctgagctcgatgtcatggcacctggcgtatctatccaaagcacgcttcctggaaacaaatacggcgcgttgaacggtacatcaatggcatctccgcacgttgccggagccgcggctttgattctttctaagcacccgaactggacaaacactcaagtccgcagctctctagaaaacaccactacaaaacttggtgattctttctactatggaaaagggctgatcaatgtacaggcggcagctcag taaaactcgagataaaaaaccggccttggccccgccggttttttat

The amino acid sequence of the BPN′-Y217L precursor protein is providedbelow. In this sequence, bold indicates the mature BPN′-Y217L protease:

mrskklwisllfalaliftmafgstssaqaagksngekkyivgfkqtmstmsaakkkdvisekggkvqkqfkyvdaasatlnekavkelkkdpsvayveedhvahayaqsvpygvsqikapalhsqgytgsnvkvavidsgidsshpdlkvaggasmvpsetnpfqdnnshgthvagtvaalnnsigvlgvapsaslyavkvlgadgsgqyswiingiewaiannmdvinmslggpsgsaalkaavdkavasgvvvvaaagnegtsgssstvgypgkypsviavgavdssnqrasfssvgpeldvmapgvsiqstlpgnkygalngtsmasphvagaaalilskhpnwtntqvrsslentttklgdsfyygkglinvqaaaq.

The amino acid sequence of the mature BPN′-Y217L protease was used asthe basis for making the variant libraries described herein:

aqsvpygvsqikapalhsqgytgsnvkvavidsgidsshpdlkvaggasmvpsetnpfqdnnshgthvagtvaalnnsigvlgvapsaslyavkvlgadgsgqyswiingiewaiannmdvinmslggpsgsaalkaavdkavasgvvvvaaagnegtsgssstvgypgkypsviavgavdssnqrasfssvgpeldvmapgvsiqstlpgnkygalngtsmasphvagaaalilskhpnwtntqvrsslentttklgdsfyygkglinvqaaaq

Each amplification reaction contained 30 pmol of each oligonucleotideand 100 ng of pAC-FNA10 template DNA (see FIG. 11) Amplifications werecarried out using Vent DNA polymerase (New England Biolabs). The PCR mix(20 ul) was initially heated at 95° C. for 2.5 min followed by 30 cyclesof denaturation at 94° C. for 15s, annealing at 55° C. for 15s andextension at 72° C. for 40s. Following amplification, left and rightfragments generated by the PCR reactions were purified by the Qiagengel-band purification kit (Valencia, Calif.) and mixed (about 200 ng ofeach fragment). Every mix contained three left fragments targeting threeadjacent codons and three right fragments targeting same codons. Thesemixes were digested with Eam1104I, ligated with T4 DNA ligase andamplified by flanking primers (P4299 CGTTGAAGAAGATCACGTAGCA, and P3246TTTATTTTATAAACTCATTCCCTGAT) to generate the full-length gene fragment.The PCR conditions were same as described above, except the extensionphase, which was carried out at 72° C. for 2 min. The resultingfragments were purified by the Qiagen gel-band purification kit(Valencia, Calif.), digested with MluI and XhoI, and cloned into theMluI/XhoI sites in the Bacillus subtilis expression plasmid pHPLT-FNA.

The BPN′-Y217L expression cassette from the pHPLT vector (Platpromoter-pre-pro-BPN′-Y217L -terminator) used has the polynucleotidesequence shown below.

GCTTTTCTTTTGGAAGAAAATATAGGGAAAATGGTACTTGTTAAAAATTCGGAATATTTATACAATATCATATGTTTCACATTGAAAGGGGAGGAAAATCGTGAAACAACAAAAACGGCTTTAGTCTAGCAAAAGGAGAGGGTAAAGAGTGAGAAGCAAAAAATTGTGGATCAGTTTGCTGTTTGCTTTAGCGTTAATCTTTACGATGGCGTTCGGCAGCACATCCTCTGCCCAGGCGGCAGGGAAATCAAACGGGGAAAAGAAATATATTGTCGGGTTTAAACAGACAATGAGCACGATGAGCGCCGCTAAGAAGAAAGATGTCATTTCTGAAAAAGGCGGGAAAGTGCAAAAGCAATTCAAATATGTAGACGCAGCTTCAGCTACATTAAACGAAAAAGCTGTAAAAGAATTGAAAAAAGACCCGAGCGTCGCTTACGTTGAAGAAGATCACGTAGCACACGCGTACGCGCAGTCCGTGCCTTACGGCGTATCACAAATTAAAGCCCCTGCTCTGCACTCTCAAGGCTACACTGGATCAAATGTTAAAGTAGCGGTTATCGACAGCGGTATCGATTCTTCTCATCCTGATTTAAAGGTAGCAGGCGGAGCCAGCATGGTTCCTTCTGAAACAAATCCTTTCCAAGACAACAACTCTCACGGAACTCACGTTGCCGGCACAGTTGCGGCTCTTAATAACTCAATCGGTGTATTAGGCGTTGCGCCAAGCGCATCACTTTACGCTGTAAAAGTTCTCGGTGCTGACGGTTCCGGCCAATACAGCTGGATCATTAACGGAATCGAGTGGGCGATCGCAAACAATATGGACGTTATTAACATGAGCCTCGGCGGACCTTCTGGTTCTGCTGCTTTAAAAGCGGCAGTTGATAAAGCCGTTGCATCCGGCGTCGTAGTCGTTGCGGCAGCCGGTAACGAAGGCACTTCCGGCAGCTCAAGCACAGTGGGCTACCCTGGTAAATACCCTTCTGTCATTGCAGTAGGCGCTGTTGACAGCAGCAACCAAAGAGCATCTTTCTCAAGCGTAGGACCTGAGCTTGATGTCATGGCACCTGGCGTATCTATCCAAAGCACGCTTCCTGGAAACAAATACGGCGCGTTGAACGGTACATCAATGGCATCTCCGCACGTTGCCGGAGCGGCTGCTTTGATTCTTTCTAAGCACCCGAACTGGACAAACACTCAAGTCCGCAGCAGTTTAGAAAACACCACTACAAAACTTGGTGATTCTTTCTACTATGGAAAAGGGCTGATCAACGTACAGGCGGCAGCTCAGTAAACTCGAGAGAGGACGGATTTCCTGAAGGAAATCCGTTTTTTTATTTTAAGCTT G

Ligation mixtures were amplified using rolling circle amplification byIllustra Templiphi kit according to the manufacturer's recommendation(GE Healthcare, Piscataway, N.J.) to generate multimeric DNA fortransformation into Bacillus subtilis. For this purpose, 1 μl of theligation mixture was mixed with 5 μl of the sample buffer, heated to 95°C. for 3 min and cooled on ice. Next, 5 μl of the reaction buffer and0.2 μl of the enzyme were added to each tube, followed by incubation at30° C. for 10 hours. Products of the rolling circle amplification werediluted 100 times and used to transform B. subtilis cells (ΔaprE, ΔnprE,amyE::xylRPxylAcomK-phleo). An aliquot of the transformation mix wasplated on LB plates containing 1.6% skim milk and 10 μg/mL neomycin andincubated overnight at 37° C. Subsequently, colonies with halos wereinoculated in 120 μl of LB media containing 10 μg/mL neomycin, coveredwith Enzyscreen lids and incubated overnight at 37° C. and 70% humiditywith vigorous shaking (250 rpm). Next morning overnight cultures werefrozen and screened later in the BMI assays as described in Test Method2.

Description

Results of experiments conducted to determine stain removal activity(microswatch assay to determine stain removal performance in laundryapplications using EMPA 116 swatches (BMI stain, CFT) at pH 8/16° C. andprotein determination by TCA precipitation (tests of properties ofinterest) of BPN′-Y217L variants are shown in Table 31-10. The resultswere obtained using the methods described in Test Method 2 with thefollowing modifications for the stain removal performance assay. Thetest detergent used was heat inactivated Tide 2× detergent (Proctor &Gamble). Heat inactivation of commercial detergent formulas serves todestroy the endogenous enzymatic activity of any protein componentswhile retaining the properties of nonenzymatic components. Heatinactivation of the detergents was performed by placing pre-weighedamounts of liquid detergent (in a glass bottle) in a water bath at 95°C. for 2 hours. The detergent was purchased from local supermarketstores. Both unheated and heated detergents were assayed within 5minutes of dissolving the detergent to accurately determine percentagedeactivated. Enzyme activity was tested by AAPF assay. As describedthroughout functionality of BPN′-Y217L variants was quantified as aperformance index (“Pi” or “PI”), which is the ratio of performance of avariant to parent protein BPN′-Y217L.

Data are shown for variants with a PI of at least 1.1 relative to FNA inBMI assays at 16C.

TABLE 31-10 Mutations and Performance Index for TCA protein and BMIActivity for BPN′ Variants PI on BMI at Mutations TCA PI pH 8 16° C.Y217L-V068C-A069G 0.4 1.2 Y217L-I079F-A098G 1.2 1.2Y217L-P086T-S101D-Q103S-V147I 0.4 1.3 Y217L-A088T-P129S-G146D 0.7 1.2Y217L-V093I-G128D-P129R 0.6 1.2 Y217L-Z096.01D-A098R 0.7 1.2Y217L-Z096.01H-A098G 0.3 1.2 Y217L-G097S-Z097.01S-A098G-A273T 0.4 1.2Y217L-A098S-D099G-G100D 0.3 1.3 Y217L-Z098.01N 0.5 1.3Y217L-D099G-Z099.01N 0.3 1.2 Y217L-D099G-Z099.01S 0.2 1.2Y217L-D099V-S101D 0.5 1.2 Y217L-Z099.01S 0.5 1.2 Y217L-G100D 0.5 1.2Y217L-S101D-Q103H 0.9 1.2 Y217L-S101G-A151V 0.4 1.2 Y217L-S101H-G102S0.7 1.2 Y217L-S101H-Q103D 1.0 1.2 Y217L-G102R-Q103C-Y104C-V192I 0.3 1.3Y217L-Q103D 1.0 1.2 Y217L-V121I-I122S-N123C 0.6 1.4 Y217L-V121L-N123C1.0 1.2 Y217L-I122S-N123S 0.6 1.3 Y217L-M124I 1.7 1.2 Y217L-M124V 1.11.2 Y217L-L126F-P129Z-S182N 1.5 1.2 Y217L-L126Y 1.0 1.3Y217L-G127S-P129D 0.7 1.3 Y217L-Z127.01N-G128S-P129S 0.6 1.2Y217L-G128H-P129Y 0.4 1.2 Y217L-G128S-P129D 0.9 1.4Y217L-G128S-P129D-S248R 0.9 1.3 Y217L-G128S-P129G 1.1 1.4Y217L-P129G-G131Z 0.6 1.2 Y217L-P129G-S130H-S132Z 0.4 1.2Y217L-P129H-G131Z 0.6 1.2 Y217L-P129L 1.0 1.2 Y217L-P129S-S130H-S132Z0.5 1.2 Y217L-P129Z 0.6 1.2 Y217L-P129Z-S130G 0.5 1.3Y217L-P129Z-S130G-G131H-S132H 1.1 1.2 Y217L-P129Z-S130H 0.6 1.2Y217L-S130V-G131D-S132I 1.0 1.2

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “40 mm” is intended to mean“about 40 mm”.

Every document cited herein, including any cross referenced or relatedpatent or application, is hereby incorporated herein by reference in itsentirety unless expressly excluded or otherwise limited. The citation ofany document is not an admission that it is prior art with respect toany invention disclosed or claimed herein or that it alone, or in anycombination with any other reference or references, teaches, suggests ordiscloses any such invention. Further, to the extent that any meaning ordefinition of a term in this document conflicts with any meaning ordefinition of the same term in a document incorporated by reference, themeaning or definition assigned to that term in this document shallgovern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

What is claimed is:
 1. A composition comprising an isolated polypeptidecomprising a variant protease, derived from the parent protease setforth by SEQ ID NO: 4, wherein said variant protease has at least 90%sequence identity to the amino acid sequence of SEQ ID NO: 4, has themutations S063T-S078N-S101A-S183T, and has protease activity.
 2. Thecomposition according to claim 1 wherein said variant protease has atotal net charge of −1, 0, or +1 relative to the protease of SEQ IDNO:2.
 3. The composition of claim 1, said composition comprising anadjunct ingredient selected from the group consisting of: a surfactant,a builder, a chelating agent, a dye transfer inhibiting agent, adispersant, one or more additional enzymes, an enzyme stabilizer, acatalytic material, a bleach activator, a hydrogen peroxide, a source ofhydrogen peroxide, a preformed peracid, a polymeric dispersing agent, aclay soil removal/anti-redeposition agent, a brightener, a sudssuppressor, a dye, a perfume, a perfume delivery system, a structureelasticizing agent, a fabric softener, a carrier, a hydrotrope, aprocessing aid, a solvent, a pigment and mixtures thereof.
 4. Thecomposition according to claim 1, said composition comprising a secondnon-immunoequivalent protease that is a subtilisin (EC 3.4.21.62), saidsecond non-immunoequivalent protease as set forth by SEQ ID NO:
 755. 5.The composition of claim 1, said composition comprising an additionalenzyme selected from the group consisting of hemicellulases,peroxidases, proteases, cellulases, cellobiose dehydrogenases,xyloglucanases, xylanases, lipases, phospholipases, esterases,cutinases, pectinases, mannanases, pectate lyases, keratinases,reductases, oxidases, phenoloxidases, lipoxygenases, ligninases,pullulanases, tannases, pentosanases, lichenases glucanases,arabinosidases, hyaluronidase, chondroitinase, laccase, amylases, andmixtures thereof.
 6. The composition of claim 1 wherein said variantprotease has a performance index from 1 to about 10 on BMI at pH 8 and60° F. when compared to the protease of SEQ ID NO:2, as defined in TestMethod 2 and/or Test Method
 3. 7. The composition of claim 1, whereinsaid composition is a detergent composition.